Cancer treatment and diagnosis

ABSTRACT

The present disclosure provides, in general, a method for selecting a therapy for treating cancer in a human subject and subsequently treating cancer in a subject, which includes isolating a cancer cell from a human subject having cancer, determining the functional activity of the innate immune regulator STimulator of INterferon Genes (STING) or the cellular nucleotidyltransferase, cyclic Guanosine Monophosphate (cGMP)—Adenosine Monophosphate (AMP) Synthase (cyclicGMP-AMP Synthase or cGAS) in the cell, and selecting a therapy for the cancer based on the functional activity of the STING or cGAS in the cell. Also provided, if the functional activity of STING and/or cGAS is determined to be defective in the cell, the therapy selected is one that is effective at killing STING-deficient and/or cGAS-deficient cancer cells, for example a therapy including administering to the subject an oncolytic virus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is (i) a Continuation of U.S. patent application Ser.No. 16/284,975, filed Feb. 25, 2019, which is (ii) a Continuation ofU.S. patent application Ser. No. 15/735,502, filed Dec. 11, 2017, whichis (iii) the U.S. National Phase Application of PCT/US2016/037288, filedJun. 13, 2016, which claims the priority benefit of (iv) U.S.Provisional Patent Application No. 62/174,374, filed Jun. 11, 2015,(i)-(iv) are herein incorporated by reference in their entireties andfor all purposes.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant Number RO1AI079336-05 awarded by the National Institute of Allergy and InfectionDiseases (NIAID). The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

This application contains, as a separate part of the disclosure, aSequence Listing in computer-readable form which is incorporated byreference in its entirety and identified as follows: Filename:STNG-01001US2_ST25.txt; Size: 2,469 bytes, created: Sep. 28, 2020.

FIELD OF THE INVENTION

The present disclosure relates generally to the fields of molecularbiology, immunology, biochemistry, cancer, and medicine. Moreparticularly, the disclosure relates to methods for diagnosis andtreatment of cancer through the use of a cellular protein.

BACKGROUND

Cancer is a leading cause of death in the United States of America andelsewhere. New treatments and diagnostics are needed to improveoutcomes.

Colorectal cancer (CRC) affects about 1.2 million people in the UnitedStates with approximately 150,000 new cases being diagnosed every year.Indeed, CRC is the third most common cause of cancer worldwide, afterlung and breast cancer, and the second leading cause of cancer death inadults (DeSantis et al., 2014). Intestine-associated malignant diseasefrequently develops from colonic epithelial cells that accumulategenetic alterations in key genes involved in the control of cell growth.Multistep genomic damage aggravated alterations can be acquired fromenvironmental factors comprising carcinogens or from genotoxic microbialpathogens including Helicobacter pylori (Arthur et al., 2014; Kim andChang, 2014; Louis et al., 2014). Such genetic amendments frequentlyinvolve activation of cell growth signaling through mutation of k-ras aswell as through mutation or epigenetic silencing of critical tumorsuppressor genes (TSGs) such as p53 and adenomatous polyposis coli(APC). Mutated TSGs such as APC can also be inherited, thus increasingthe risk of CRC significantly (Fearon, 2011).

Orally administered carcinogens such as the DNA-adduct formingazoxymethane (AOM) induce genomic changes in gastrointestinal epithelialcells, an event which can trigger the activation of DNA damage response(DDR) pathways. While these responses involve repairing DNA breaks andeliminating base mismatches, they can also include activating theproduction of pro-inflammatory cytokines which alerts the immunesurveillance system to the damaged area and facilitates wound repair.For example, using murine models, it has been demonstrated that theadministration of AOM followed by inflammatory drug dextran sulphatesodium (DSS) can cause epithelial cells to produce IL-1 (3 and IL-18which becomes processed by the inflammasome, a multiprotein complexcomprising nucleotide-binding oligomerization-domain protein likereceptors (NLRB) such as NLRP3 and NLRP6 as well as apoptotic speckprotein containing a CARD (ASC/PYCARD) and caspase-1, for secretion(Arthur et al., 2012; Elinav et al., 2011). IL-18, for example, can bindto colonic dendritic cells and signal through MyD88 to prevent theproduction of growth inhibitory IL-22 binding protein (IL-22BP), whichenables unrestricted IL-22 to stimulate tissue repair (Huber et al.,2012; Salcedo et al., 2010). Thus, mice defective in keyinflammasome-associated molecules such as ASC or caspase-1 aresusceptible to carcinogen induced colitis-associated cancer (CAC).Similarly, loss of key adaptor molecules such as MyD88, required forIL1-R signaling are susceptible to AOM/DSS induced CAC. Plausibly,unrepaired lesions enable the infiltration of microbes with heightenedgenotoxic aptitude that can chronically aggravate inflammatory processesand the production of DNA damaging radical oxygen species (ROS).

While the inflammasome has been shown to be important for processingproinflammatory cytokines such as IL1β and IL-18, it remained to befully clarified how such wound repair proteins become transcriptionallyactivated in response to actual genomic damage. However, it has recentlybeen shown that mice lacking the innate immune regulator STING(stimulator of interferon genes) are also sensitive to AOM/DSS-inducedCAC (Ahn et al., 2015). STING resides in the endoplasmic reticulum (ER)of hematopoietic cells as well as endothelial and epithelial cells andcontrols the induction of numerous host defense genes, such as type IIFN as well as pro-inflammatory genes including ILH3 in response to thedetection of cyclic dinucleotides (CDNs) such as cyclic-di-AMP(c-di-AMP) generated from intracellular bacteria (Ichikawa and Barber,2008; Woodward et al., 2010). STING is also the sensor for CDNs producedfrom a cellular nucleotidyltransferase referred to as cGAS (cyclicGMP-AMP synthase, also referred to as Mab-21 Domain-Containing Proteinand C6orf150) (Sun et al., 2013). Cytosolic DNA species which canconstitute the genome of invading pathogens such as HSV-1, or plausiblyself-DNA leaked from the nucleus can bind to cGAS to generatenon-canonical cGAMP containing one 2′-5′ phosphodiester linkage and acanonical 3-5′ linkage (c[G(2′,5′)pA(3′,5′)p]). The STING pathway mayrecognize damaged DNA during early response to intestinal damage and maybe essential for invigorating tissue repair pathways involving IL1β andIL-18 (Ahn et al., 2015). STING has also been recently reported to playan essential role in dendritic cell recognition of dying tumor cells andthe priming of anti-tumor cytotoxic T-cell (CTL) responses (Corrales etal., 2015; Woo et al., 2014). Thus, while loss of STING may facilitatetumorigenesis through preventing wound repair and by preventing theproduction of tumor specific CTLs, the effectiveness of STING signalingin human tumors remains unknown.

SUMMARY

It is reported herein that STING mediated innate immune signaling islargely impaired in human colon cancers as well as many other types ofhuman cancers. In many instances, this was achieved through silencingSTING and/or synthase cGAS expression through epigenetichypermethylation processes. The findings suggest that STING pathway mayhave a major function in suppressing colon tumorigenesis and that theinhibition of STING function in this pathway may be selectivelysuppressed during cancer development. Additionally, it is discoveredthat defects in STING signaling renders cancer cells more susceptible tooncolytic viral infection. Therefore, the examination of STING activityin cancers may lead to development of assays that will shed light intothe outcome of select cancer therapies.

It was discovered that the cellular protein STING, which controls innateimmune responses to cytoplasmic DNA produced by DNA damaging agents orDNA viruses, is defective in a wide variety of cancer cells. Defects inSTING signaling may help tumor cells evade purging by the immune systemand constitute a common mechanism of tumorigenesis. Examining STINGexpression in tumors allows predicting disease outcome and provides acrucial prognostic marker in predicting responses to select anti-tumortherapies. Disclosed herein are experiments showing that mice-deficientin STING (STING knockout or SKO) are prone to colitis associated cancer(CAC) induced by DNA-damaging and inflammatory agents. SKO miceharboring tumors exhibited low levels of tumor suppressive IL22 bindingprotein (IL22-BP) compared to normal mice, a cytokine important forpreventing colon-related tumorigenesis. Analysis of human colon cancercells and a variety of other cancer cells such as melanoma indicatedwidespread defects in STING signaling which frequently involved completeloss of STING and/or cyclic GMP-AMP synthase (cGAS), a synthase thatgenerates STING-activating cyclic dincucleotides (CDN's). Such tumorcells were highly susceptible to viral oncolytic therapy.

Disclosed herein are methods for selecting a therapy for treating cancerin a mammalian (e.g., human) subject, and treating a subject with theselected therapy. One such method includes the steps of: isolating asample from a human subject having cancer; determining the functionalactivity of STING and/or cGAS in the sample; selecting a therapy for thecancer based on the functional activity of the STING and/or cGAS in thesample, and treating a subject with the selected therapy. Alsocontemplated is the measurement of levels of IL-22BP's suppression ofIL-22, as well as cellular levels of IL-1 (3, IL-18 and IL-22. Adecrease in levels of IL-1 (3, IL-18, IL-22 and IL-22BP may beindicative of defective STING or cGAS signaling.

In various embodiments, the sample is a body fluid, cell, tissue sample,biopsy, tissue print, skin, hair, a soluble fraction of a cellpreparation, or media in which cells were grown. It is contemplated thatthe body fluid is blood, urine, plasma, saliva, or cerebrospinal fluid.

If the functional activity of STING and/or cGAS is determined to bedefective in the sample, the therapy selected is one that is effectiveat killing STING-deficient and/or cGAS-deficient cancer cells (e.g.,therapy including administering to the subject an oncolytic virus suchas one having a dsDNA genome, including herpes simplex virus (HSV),Varicella Zoster virus (VZV), or vaccinia virus (VV)). Exemplary virusfamilies that have dsDNA genomes include, but are not limited to,Alloherpesviridae, Herpesviridae, Malacoherpesviridae, Lipothrixviridae,Rudiviridae, Adenoviridae, Ampullaviridae, Ascoviridae, Asfarviridae,Baculoviridae, Bicaudaviridae, Clavaviridae, Corticoviridae,Fuselloviridae, Globuloviridae, Guttaviridae, Hytrosaviridae,Iridoviridae, Marseilleviridae, Mimiviridae, Nudiviridae, Nimaviridae,Pandoraviridae, Papillomaviridae, Phycodnaviridae, Plasmaviridae,Polydnaviruses, Polyomaviridae, Poxviridae, Sphaerolipoviridae,Tectiviridae and Turriviridae.

In various embodiments, the examination of STING-signaling is a usefulprognostic marker for whether HSV1 or other viral based anti-cancertherapies will be efficacious for the treatment of malignant disease.

In the methods described herein, the subject can be one that has failedat least one chemotherapy regimen (e.g., one that includes administeringto the subject an agent which causes DNA mutations) and the step ofdetermining the functional activity of STING in the cell can includeanalyzing the amount of cGAS in the cell.

In various embodiments, it is contemplated that the selected therapy,e.g., an oncolytic virus, is administered in conjunction with a secondtherapeutic agent, such as a chemotherapeutic agent. Exemplarychemotherapeutic agents are described below in the Detailed Description.

Also disclosed herein is a method for treating a cancer in a mammalian(e.g., human) subject that includes the steps of: determining thefunctional activity of STING in a cell making up the cancer; and if thecell does not have defective STING activity, administering a cancertreatment to the subject that does not cause DNA mutation.

Further disclosed herein is a method for treating cancer in a mammalian(e.g., human) subject which includes the steps of: isolating a samplefrom a human subject having cancer; determining the susceptibility ofthe cancer to being killed by an oncolytic virus in vitro; and if thecancer is susceptible to being killed in this manner, administering anoncolytic virus to the subject. In various embodiments, the step ofdetermining the functional activity of STING in the sample comprisesanalyzing the amount of cGAS in the cell. Also contemplated is themeasurement of levels of IL-22BP's suppression of IL-22, as well ascellular levels of IL-1 β, IL-18 and IL-22. A decrease in levels of IL-1(3, IL-18, IL-22 and IL-22BP may be indicative of defective STING orcGAS signaling.

In various embodiments, the cancer is colorectal cancer,colitis-associated cancer or melanoma. Additional exemplary cancerscontemplated for treatment herein are set out in the detaileddescription.

In various embodiments, measurement of the presence or absence ofSTING/cGAS expression is predictive of the response of patients withcertain cancers to viral oncolytic therapy. In various embodiments,measurement of response may be carried out using fluorescence in situhybridization, and analysis of STING and/or cGAS protein or RNAexpression, to predict the outcome to oncolytic viral therapy dependingon the presence or absence of cGAS or STING.

Provided herein is a method for treating cancer comprising administeringa viral oncolytic therapy to a subject, determining the level of STINGor cGAS in the subject, wherein a decrease in STING or cGAS activity ispredictive of a positive outcome of oncolytic therapy, and i) if levelsof STING or cGAS in the subject are low, continuing oncolytic therapy;or ii) if STING or cGAS levels are normal or partially active,discontinuing viral oncolytic therapy and/or administering a secondagent that can increase STING levels in the subject in order to improvethe outcome of the viral oncolytic therapy.

In various embodiments, the viral oncolytic therapy comprisesherpesvirus, VZV or vaccinia virus.

In various embodiments, the determining comprises obtaining a samplefrom the subject and measuring levels of STING, cGAS, or otherbiomarkers contemplated herein (e.g., IL-18, IL-22, IL-22BP, IL-1β,IFNβ, type I IFN) in the sample. It is contemplated that the sample is abody fluid, such as blood, urine, plasma, saliva, or cerebrospinalfluid; a cell; a tissue; a tissue print; a fingerprint, skin or hair;and the like; a soluble fraction of a cell preparation, or media inwhich cells were grown; a chromosome, an organelle, or membrane isolatedor extracted from a cell; genomic DNA, RNA, or cDNA, polypeptides, orpeptides in solution or bound to a substrate.

In various embodiments, an immune response in the cancer that is lackingSTING activity or cGAS activity is enhanced by administration of anoncolytic virus. In one embodiment, the immune response includesmodulation of T cell activity, modulation of dendritic cell activity, ormodulation of immune cytokines.

In various embodiments, the therapy results in increased tumor celldeath and/or retarded tumor growth in a subject.

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the invention and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment”, “some embodiments”, “certain embodiments”, “furtherembodiment”, “specific exemplary embodiments”, and/or “anotherembodiment”, each of these types of embodiments is a non-limitingexample of a feature that is intended to be combined with any otherfeature, or combination of features, described herein without having tolist every possible combination. Such features or combinations offeatures apply to any of the aspects of the invention. Where examples ofvalues falling within ranges are disclosed, any of these examples arecontemplated as possible endpoints of a range, any and all numericvalues between such endpoints are contemplated, and any and allcombinations of upper and lower endpoints are envisioned.

Unless otherwise defined, all technical terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this invention belongs. Although methods and materials similar orequivalent to those described herein can be used in the practice ortesting of the present invention, suitable methods and materials aredescribed below. All publications, patents, and patent applicationsmentioned herein are incorporated by reference in their entirety. In thecase of conflict, the present specification, including definitions willcontrol. In addition, the particular embodiments discussed below areillustrative only and not intended to be limiting.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E show activation of STING-dependent genes by azoxymethane(AOM) (FIG. 1A) Gene array analysis of Wild type (WT) and STINGdeficient (SKO) mouse embryonic fibroblasts (MEFs) treated with AOM at0.14 mM for 8 hours (Left) and 1,2-dimethylhydrazine (DMH) at 1 mM for 8hours (Right). Highest variable genes are shown. Rows representindividual genes; columns represent individual samples. Grayscaleindicates transcript levels below, equal to, or above the mean. Scalerepresents the intensity of gene expression (log 2 scale ranges between−2.4 and 2.4). (FIG. 1B) qPCR analysis of Cxcl10 and Ifit3 in MEFstreated with AOM and DMH same as FIG. 1A. (FIG. 1C) qPCR analysis ofCxcl10 in Human epithelial cell (FHC) treated with AOM and DMH at 1 mMfor 24 hours. (FIG. 1D) FHC cells were transfected with STING or controlsiRNA for 72 hours followed by AOM and DMH treatment same as FIG. 1C,and were then subjected to Cxcl0 mRNA expression (Left). STINGexpression level after siRNA treatment was determined by qPCR (Right).Data is representative of at least two independent experiments. Errorbars indicate s.d. *; p<0.05, Student's t-test. (FIG. 1E) STINGImmunohistochemistry staining of the colon tissue from WT and SKO mice(Left) and Human. All images were shown at original magnification, 200×.

FIGS. 2A-2F show loss of STING renders mice susceptible to CAC: (FIG.2A) Schematic representation of AOM/DSS induced colitis model. WT (n=7)and SKO (n=7) mice were intravenously injected with AOM on Day 1followed by 7 d administration of dextran sodium sulfate (DSS) indrinking water for four DSS cycles. Normal drinking water was used forcontrol group. (FIG. 2B) Representative photographs of macro-endoscopiccolon tumors (Left) and H&E staining (Right) of WT (n=7) and SKO (n=7)mice either AOM/DSS treated or normal water treated. Number of polyps(FIG. 2C) and inflammation score (FIG. 2D, 0: Normal to 3: most severe)from FIG. 2B. (FIG. 2E) Gene array analysis of colon tissue from WT andSKO mice treated same as FIG. 2A (grayscale representation). (FIG. 2F)Gene array analysis of colon tissue from WT and SKO mice treated same asFIG. 2A (intensity of gene expression). Highest variable gene lists areshown (Right table). Rows represent individual genes; columns representindividual samples. Grayscale indicate transcript levels below, equal to(black), or above the mean. Scale represents the intensity of geneexpression (log 2 scale ranges between −2.4 and 2.4).

FIGS. 3A-3C show suppression of IL22BP expression in STING-deficientmice: (FIG. 3A) Fold changes from gene array analysis of IL18 in WT andSKO MEFs administrated with 4 ug/ml of dsDNA90 and IFNβ for 8 hours(Left). qPCR analysis of IL18 in WT and SKO MEFs transfected with 4μg/ml of dsDNA90 and cyclic-di-GMP-AMP (cGAMP) for 8 hours (Middle).qPCR analysis of IL18 in bone marrow derived dendritic cells (BMDCs)from WT and SKO mice. BMDCs were treated with 1 mM of AOM and 1 mM ofDMH for 8 hours (Right). (FIG. 3B) Schematic representation, bodyweight, and qPCR analysis of IL18, IL22 bp and IL22 from WT and SKOcolon during one cycle of DSS administration for 5 days followed by 2days of normal water. (FIG. 3C) qPCR analysis of IL18, IL22 bp and IL22in WT and SKO colon tissue from FIG. 2. Data is representative of atleast two independent experiments. Error bars indicate s.d. *; p<0.05,Student's t-test.

FIGS. 4A-4G show cytosolic DNA induced innate immune signaling wasmostly defective in human colon cancer cells: (FIG. 4A) Immunoblot ofSTING in a series of human colon cancer cell lines of various type.hTERT and normal human colon epithelial cell line, FHC, were included aspositive controls. 20 μg of total protein/per lane was loaded andanalyzed by rabbit anti STING polyclonal antibody. β-actin was used asloading control. (FIG. 4B) ELISA analysis of human Interferon 13production in the media of cells, same as in (FIG. 4A) following polyL⋅Cor dsDNA90 transfection at 3 μg/ml for 16 hours. Lipofectamine 2000alone was used as mock transfection. (FIG. 4C) Cells, same as in A, wereeither mock transfected or transfected with polyL⋅C or dsDNA90 at 3μg/ml for 3 hours. Total RNA was extracted and analyzed by qPCR for IFNβexpression. (FIG. 4D) RNA, same as in FIG. 4C, was analyzed by qPCR forCXCL10 expression. (FIG. 4 G) RNA, same as in FIG. 4C, was analyzed byqPCR for IL1β expression. (FIG. 4E) Gene array analysis of normal orcolon cancer cells mock transfected or transfected with 3 μg/ml dsDNA90for 3 hours. Highest variable genes are shown. Rows represent individualgenes; columns represent individual samples. Grayscale legend indicatestranscript levels below, equal to, or above the mean. Scale representsthe intensity of gene expression (log 10 scale ranges between −3 and 3).(FIG. 4F) List of highest variable genes shown in FIG. 4E as well astheir fold induction value following dsDNA90 stimulation. Data isrepresentative of at least two independent experiments. Error barsindicate s.d.

FIGS. 5A-5F show STING activation and cytosolic DNA pathway in coloncancer cells were mostly defective: A series of colon cancer cells aswell as normal cell controls were either mock transfected or transfectedwith dsDNA90 at 3 μg/ml for 3 hours, and were analyzed byImmunofluorescence Microscopy for STING translocation (FIG. 5A), IRF3translocation (FIG. 5B), and p65 translocation (FIG. 5C). (FIG. 5D)Cells, same as above, were either mock transfected or transfected withdsDNA90 at 3 μg/ml for indicated time periods followed by immunoblotanalysis for STING phosphorylation as well as phosphorylation of TBK1,IRF3 and p65. β-actin was used as loading control. (FIG. 5E) Cells, sameas above, were analyzed by qPCR for cGAS expression. (FIG. 5F) Cellsthat have undetectable level of cGAS in E were treated with 1045-azacytidine for 7 days, followed by qPCR analysis for GAS expression.Data is representative of at least two independent experiments. Errorbars indicate s.d.

FIGS. 6A-6D show HSV1 viral production is more effective in colon cancercells that have defected STING innate immune pathway. (FIG. 6A) A seriesof colon cancer cells as well as normal cell controls were infected withHSV-luc at M.O.I. 1 or 5 for 24 hours. Cells were then lysed andanalyzed for luciferase activity. Data is representative of at least twoindependent experiments. Error bars indicate s.d. (FIG. 6B) Cells, sameas in FIG. 6A, were infected with HSV-luc at M.O.I. 10 for 6 hours.Total RNA was then extracted, followed by qPCR analysis of IFNβproduction. (FIG. 6C) Same RNA from FIG. 6B was analyzed by qPCRanalysis for CXCL10 production. (FIG. 6D) Cells, same as in FIG. 6A,were infected with HSV1γ34.5 deletion mutant at M.O.I. 1 for 6 hoursfollowed by qPCR analysis of IFNβ production. Data is representative ofat least two independent experiments. Error bars indicate s.d.

FIGS. 7A-7B show gene expression fold changes of Illumina array shown inFIG. 1A.

FIGS. 8A-8C show fluorescence microscopy analysis (related to FIG. 1) ofDAPI staining in WT and SKO MEFs treated with 3 mM of AOM of 3 mM of DMH(FIG. 8A) and anti-dsDNA staining and the ration of cytoplasm to nucleus(FIG. 8B) in Human normal colon epithelial cells (FHC) treated with 3 mMof AOM or 3 mM of DMH for 48 hours. (FIG. 8C) Immunofluorescencemicroscopy analysis of FHC treated with AOM and DMH sane as FIG. 8A for48 hours using p65 or IRF3 antibody. Images shown at originalmagnification, 160×.

FIGS. 9A-9B show (FIG. 9A) the number of polyps and (FIG. 9B) shows theinflammation score from FIG. 2A-2F.

FIGS. 10A-10B show primary MEF cells lacking STING and/or p53 weretransduced with retrovirus encoding human H-Ras 12V or human c-Myc.After drug selection with puromycin and hygromycin, the cells werecultured in soft agar. After 14 days, colonies were photographed (FIG.10A) and colony numbers in one well (n=3) were counted (FIG. 10B). Errorbars indicated standard deviation.

FIG. 11. IL18 promoter region contains binding sites for multiple innateimmune gene transcription factors. Putative transcription factor bindingsites in the IL18 gene promoter is listed and highlighted.

FIG. 12. Shown is a summary of STING signaling pathway in colon cancercell lines.

FIGS. 13A-13D show human colon cancer cells (SW480 and HT116) as well ashTERT cells were treated with 1 uM 5azacytidine for 7 days, followed bydsDNA90 transfection at 3 μg/ml for 3 hours. Total RNA was extracted andanalyzed by qPCR for cGAS (FIG. 13A) and IFNβ (FIG. 13B) expression.(FIG. 13C) cGAS production is deregulated in many colon cancers. cDNAfrom 5 normal human colon tissues and 43 human colon cancers of variousstages were analyzed by qPCR for cGAS expression. (FIG. 13D) Immunoblot(upper) and qPCR analysis (lower) of cGAS expression in normal and humancolon cancer cells same as above.

FIGS. 14A-14C show (FIG. 14A), [[I]]immunoblot of STING in varioustransformed or cancer derived human cell lines. HUVEC was a positivecontrol. FIG. 14B, Northern blot analysis of STING mRNA expression incell lines as in FIG. 14A. HUVEC was a positive control. FIG. 14C, ELISAanalysis of IFNβ production in the media of cells transfected with 3μg/ml polyL⋅C or dsDNA90 or mock transfected for 16 hours. PASMC,NHDF-ad and hTERT were included as positive controls.

FIGS. 15A-15E show cGAS expression is suppressed in many human coloncancer cell lines and can be partially recapitulated through DNAdemethylation. FIG. 15A, Immunoblot (upper) and qPCR analysis (lower) ofcGAS expression in normal and human colon cancer cells same as above.FIG. 15B, qPCR analysis of cGAS expression in cGAS negative colon celllines mock treated or treated with 104 5-Azacytidine (SAZADC) for 5days. FIG. 15C, Immunoblot analysis of STING signal activation in cells(selected from FIG. 15B) mock treated or treated with 104 5-Azacytidine(SAZADC) for 5 days, followed by dsDNA90 transfection at 3 μg/ml forindicated time periods. FIG. 15D, Immunofluorescence Microscopy analysisof IRF3 translocation in SW480 and HT116 cells treated with SAZADC sameas above followed by dsDNA transfection at 3 μg/ml dsDNA90 for 3 hours.Original magnification, 1260×. FIG. 15E, IFNβ qPCR analysis of cells(same as in FIG. 15C) treated with 5AZADC same as above followed bydsDNA transfection at 3 μg/ml dsDNA90 for 3 hours. Error bars indicates.d. *, p<0.05; **, p<0.01; ***, p<0.001; Student's t-test.

FIGS. 16A-16F show STING signal defect leads colon cancer cells moresusceptible to DNA virus infection. (FIG. 16A), Cells (same as in FIG.4A-4F) were infected with HSV1γ34.5 at M.O.I. 5 for 1 hour and humanIFNβ induction was analyzed by qPCR 3 hours post infection. FIG. 16B,normal human hTERT cells and selected human colon cancer cell lines(cGAS positive: SW1116, HT29; cGAS negative: SW480, HT116) were infectedwith HSV1γ34.5 at indicated M.O.I. for 1 hour, and titration ofHSV1γ34.5 was analyzed by standard plaque assay in Vero cells 24 hourslater. FIG. 16C, Cells (same as in FIG. 16B) were infected withHSV1γ34.5 at M.O.I. 1 for 1 hour, and cell viability was analyzed bytrypan blue staining 24 hours and 48 hours later. FIG. 16D, Cells (sameas in FIG. 16A) were infected with HSV1-Luc at indicated M.O.I. for 1hour, and luciferase activity was analyzed 24 hours later. FIG. 16E,Colon Cancer cells were infected with Vaccinia Virus at M.O.I. 100 andanalyzed by qPCR for IFNB expression 3 hours post infection. FIG. 16F,cells same as FIG. 16E were analyzed by qPCR for CXCL10 expression.Error bars indicate s.d.

FIGS. 17A-17H show RNA in situ hybridization analysis of STING and cGASin human colon cancer cell lines and colon cancer tissue microarray.FIG. 17A, RNA fluorescence in situ hybridization (RNA FISH) analysis ofSTING and cGAS expression in normal and human colon cancer cell lines.Images are shown at 1260×. FIG. 17B, RNA FISH analysis of STING and cGASexpression in SW480 and HT116 mock treated or treated with 1 i.t.M 5AZADC for 5 days. Images are shown at 1260×. FIG. 17C, Quantitation ofSTING and cGAS RNA copy number in FIG. 17A. FIG. 17D, Quantitation ofcGAS RNA copy number in FIG. 17B. FIG. 17E, STING and cGAS expression informalin-fixed paraffin-embedded (FFPE) normal and human colon cancercell lines were analyzed by Chromogenic RNA in situ hybridization (RNACISH). Quantitation of STING and cGAS RNA copy number are shown in bargraph. Error bars indicate s.d. FIG. 17F, representative images of STINGand cGAS RNA CISH analysis are shown at 600×. FIG. 17G, RNA CISHanalysis of STING and cGAS expression in a FFPE human colon cancertissue microarray. A total of 12 normal and 80 cancer tissues wereanalyzed and number of tissue that are detected with STING and/or cGASare summarized in the table. FIG. 17H, Representative images of RNA CISHin FIG. 17G are shown at 400×.

FIGS. 18A-18E show increased HSV1γ34.5 oncolytic effect was observed incolon cancer cells with impaired STING signal in vivo. FIG. 18A, Schemeof HSV1γ34.5 treatment on xenograft tumor in nude mice. The indicatedxenograft tumors (SW116, FIG. 18B; HT29, FIG. 18C; SW480, FIG. 18D;HT116, FIG. 18E) were generated in the right flank of nude Balb/c mice.When tumors had reached approximately 0.5 cm in diameter, tumors wereinjected every other day a total of three times (arrows) with 1E7 PFUHSV1γ34.5 in 50 μl PBS (N=7) or 50 μl PBS only (N=3) and tumor growthmeasured every other day. Statistical analysis was carried out comparingthe two treatment groups at the last time point using the unpairedStudent's t-test. P values are as indicated.

FIG. 19 shows dsDNA90 transfection efficiency into colon cancer celllines monitored with FITC-dsDNA90 3 hours post Lipofectamine 2000transfection under fluorescent microscopy. Images shows at 400×.

FIGS. 20A-20D show normal and colon cancer cell cells were treated withnon-specific siRNA (si-NT) or STING siRNA (si-STING) for 3 days followedby dsDNA90 transfection at 3 μg/ml for 3 hours. Cells were then analyzedfor STING siRNA efficiency by immunoblot (FIG. 20A) and by qPCR for IFNβexpression (FIG. 20B) and CXCL10 expression (FIG. 20C). FIG. 20D, cellswere similarly treated with siRNA as above followed by HSVγ34.5infection at MOI 5 for 3 hours. Cells were then analyzed by qPCR forIFNβ expression.

FIGS. 21A-21C show (FIG. 21A), schematic representation of CpG islandslocated in the proximal promoter regions of cGAS. FIG. 21B, Bisulfitesequencing analysis of cGAS promoter region. Each box represents one CpGdinucleotide located within the promoter region indicated by theposition marker at the bottom. Grayscale compares methylated,unmethylated and not sequenced. FIG. 21C, colon cancer cells weretreated with SAZADC (DNA methyltransferase inhibitor), SAHA (histonedeacetylase inhibitor) and BIX01294 (histone-lysine methyltransferaseinhibitor) at 104 for 5 days. cGAS expression was then examined by qPCR.Error bars indicate s.d.

FIG. 22. Normal and colon cancer cells were treated with AOM or DMH at15 mM for 20 hours. IFNβ induction was analyzed by qPCR. STING, IRF3 andNF-κB translocation was examined: +, translocation; −, no translocation.

FIGS. 23A-23C show. (FIG. 23A), immunoblot of STING in varioustransformed or cancer derived human cell lines. HUVEC was used aspositive control. FIG. 23B, Northern blot analysis of STING mRNAexpression in cell lines. FIG. 23C, ELISA analysis of IFNβ production inthe media of cells transfected with 3 μg/ml polyL⋅C or dsDNA90 or mocktransfected for 16 hours. PASMC, NHDF-ad and hTERT used as positivecontrol.

FIG. 24 shows sequencing of STING in colon cancer cell lines.

FIG. 25 shows sequencing of cGAS in colon cancer cell lines.

FIGS. 26A-26D show STING expression is suppressed and dsDNA inducedinnate immune activation is impaired in majority of human melanoma celllines. FIG. 26A, hTERT fibroblasts, normal human epidermal melanocytes(HEMa) and a series of human melanoma cell lines were analyzed for STINGexpression by immunoblot (top) and cGAS expression by qPCR (bottom).FIG. 26B, ELISA analysis of human Interferon β production in the mediaof cells (same as A) transfected with 3 μg/ml polyIC or dsDNA90 or mocktransfected for 16 hours. qPCR analysis of human CXCL10 (FIG. 26C) andIFNβ (FIG. 26D) induction in cells (same as FIG. 26A) transfected with 3μg/ml dsDNA90 or mock transfected for 3 hours.

FIGS. 27A-27D show dsDNA induced STING signaling pathway is defective inmajority of human melanoma cell lines. Immunofluorescence Microscopyanalysis of STING translocation (FIG. 27A), IRF3 translocation (FIG.27B) and p65 translocation (FIG. 27C) in normal and human melanoma celllines transfected with 3 μg/ml dsDNA90 or mock transfected for 3 hours.Original magnification, 1260×. Bar size, li.t.m. FIG. 27D, Immunoblotanalysis of STING signal activation in cells (same as above) transfectedwith 3 μg/ml dsDNA90 for indicated time periods.

FIGS. 28A-28C show RNA in situ hybridization and immunohistochemistryanalysis of STING and cGAS in human melanoma cell lines. FIG. 28A, RNAfluorescence in situ hybridization (RNA FISH) analysis of STING and cGASexpression in normal and human melanoma cell lines. Representativeimages are shown at 1260×. Bar size, 500 nm. Quantitation of STING andcGAS RNA copy number are shown in bar graph. FIG. 28B, Chromogenic RNAin situ hybridization (RNA CISH) analysis of STING and cGAS expressionin formalin-fixed paraffin-embedded (FFPE) normal and human melanomacell lines. Representative images are shown at 600×. Bar size, li.t.m.Quantitation of STING and cGAS RNA copy number are shown in bar graph.FIG. 28C, Immunohistochemistry analysis of STING and cGAS expression inmelanoma cells. Images were shown at 400×. Bar size, 20 μm.

FIG. 29. STING and cGAS expression were suppressed in high percentage ofhuman melanomas. Immunohistochemistry analysis of STING and cGAS inhuman melanoma tissue microarray containing normal human epidermal andhuman melanoma tissues. Representative images of normal human epidermaland human melanoma tissues stained for STING and cGAS. Images are shownat 400×. Bar size, 50i.t.m. STING and cGAS expression status issummarized and shown in bottom panel.

FIGS. 30A-30G show DNA demethylation partially recapitulated STING andcGAS expression in human melanoma cell lines. FIG. 30A, qPCR analysis ofcGAS expression in indicated human melanoma cells mock treated ortreated with 1 i.t.M 5-Azacytidine (5AZADC) for 5 days. FIG. 30B,Immunoblot analysis of STING in indicated human melanoma cells treatedsame as above. FIG. 30C, RNA FISH analysis of STING and cGAS in cells(same as above) treated with 5AZADC same as above. Representative imagesare shown at 1260×. Bar size, 400 nm. qPCR analysis of IFNβ (FIG. 30D)and CXCL10 (FIG. 30E) in cells (same as above) treated with 5AZADCfollowed by dsDNA transfection at 3 μg/ml dsDNA90 for 3 hours.Immunofluorescence Microscopy analysis of IRF3 translocation (FIG. 30F)and STING translocation (FIG. 30G) in indicated cells treated same as inFIG. 30D. Representative images are shown at 1260×. Bar size, 500 nm.

FIGS. 31A-31D show STING signal defect leads melanoma cells moresusceptible to HSV] infection. Cells (same as in FIG. 1) were infectedwith HSV1γ34.5 at M.O.I. 5 for 1 hour and human IFNβ (FIG. 31A) andCXCL10 (FIG. 31B) induction was analyzed by qPCR 3 hours post infection.FIG. 31C, normal human hTERT cells and selected human melanoma celllines were infected with HSV1γ34.5 at indicated M.O.I. or M.O.I. 10 for1 hour, and titration of HSV1γ34.5 was analyzed by standard plaque assayin vero cells 24 hours later. FIG. 31D, Cells (same as in FIG. 31C) wereinfected with HSV1γ34.5 at M.O.I. 10 for 1 hour, and cell viability wasanalyzed by trypan blue staining 24 hours and 48 hours later.

FIGS. 32A-32D show increased HSV1γ34.5 oncolytic effect was observed inmelanoma xenografts with impaired STING signal in vivo. FIG. 32A, A375;FIG. 32B, SK-MEL-5; FIG. 32C, RPMI7951; and FIG. 32D, SK-MEL-3 melanomaxenografts were generated in the right flank of nude Balb/c mice. Whentumors had reached approximately 0.5 cm in diameter, tumors wereinjected every other day a total of three times (arrows) with 1E7 PFUHSV1γ34.5 in 50 μl PBS or 50 μl PBS only and tumor growth measured everyother day. Statistical analysis was carried out comparing the twotreatment groups at the last time point using the unpaired Student'st-test. P values are as indicated.

DETAILED DESCRIPTION

The present disclosure provides methods for selecting a cancer treatmenttherapy which involves assessing a cell of the cancer for STING activityand treating cancer with an indicated therapy. The below describedembodiments illustrate representative examples of these methods.Nonetheless, from the description of these embodiments, other aspects ofthe invention can be made and/or practiced based on the descriptionprovided below.

General Methods

Methods involving conventional immunological and molecular biologicaltechniques are described herein. Immunological methods are generallyknown in the art and described in methodology treatises such as CurrentProtocols in Immunology, Coligan et al., ed., John Wiley & Sons, NewYork. Techniques of molecular biology are described in detail intreatises such as Molecular Cloning: A Laboratory Manual, 2nd ed., vol.1-3, Sambrook et al., ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 2001; and Current Protocols in Molecular Biology,Ausubel et al., ed., Greene Publishing and Wiley-Interscience, New York.General methods of medical treatment are described in McPhee andPapadakis, Current Medical Diagnosis and Treatment 2010, 49^(th)Edition, McGraw-Hill Medical, 2010; and Fauci et al., Harrison'sPrinciples of Internal Medicine, 17^(th) Edition, McGraw-HillProfessional, 2008.

An analysis of the function of STING in colon cancer cells was conductedand found that STING was frequently expressed but STING function wasablated in approximately 86% of cells analyzed (n=12). However, the cGASwas not detectable in 30-50% of cells analyzed. In colon cancer cellslacking cGAS, STING function was completely ablated. In cancer coloncells with detectable cGAS, STING function was dramatically reduced. Itwas also noted that STING and cGAS were gone in a variety of othercancers including melanoma.

The innate immune system provides the first line of defense againstpathogen infection though can also influence pathways that can controltumorigenesis. For example, it is known that the cellular adaptor MyD88(Myeloid differentiation primary response gene 88) that facilitatesToll-like receptor (TLR) and IL-1 receptor (IL-1R) signaling pathway inthe innate immune response can regulate tumorigenesis through control ofNF-κB activation, cytokine secretion and inflammatory responses. Micelacking MyD88 are susceptible to colitis-associated carcinogenesis (CAC)induced by the drugs azoxymethane (AOM) and dextran sulfate sodium(DSS). In this situation, MyD88 exerts a protective effect in part byfacilitating the production of IL-18, in epithelial cells, whichdownregulates dendritic cell production of the IL-22 binding protein(IL-22-BP). IL-22-BP suppresses the function of IL-22 which is producedfrom innate lymphoid cells in response to cellular/tissue damage andwhich potently stimulates the proliferation of intestinal epithelialcells.

Azoxymethane (AOM) is the metabolite of 1,2-dimethylhydrazine (DMH) andis converted to methylazoxymethanol (MAM) which mediatesO-methyl-guanine formation to trigger DNA damage responses. A singleinjection of AOM into mice, followed by administration of theinflammatory agent dextran sulfate sodium (DSS) via drinking waterinduces almost 100% colon cancer. It was previously demonstrated thatthe cellular protein STING (stimulator of cellular genes) facilitatescytosolic DNA-triggered innate immune signaling pathways, independent ofToll-Receptor 9 or the DNA sensor AIM II. In humans, STING is a 348amino acid endoplasmic reticulum (ER) associated molecule predominantlyexpressed in epithelial cells as well as cells of the hematopoieticlineage, that has been shown to play a key role in triggering innateimmune signaling pathways in response to infection by viruses such asherpes simplex virus 1 (HSV1), and even bacteria. STING has also beenshown to be responsible for triggering vascular and pulmonary syndrome,self-DNA-induced inflammatory diseases such as Aicardi Goutieressyndrome (AGS) perhaps forms of severe systemic lupus erythematosus(SLE). STING may be associated with dsDNA-species directly and is highlyactivated by cyclic dinucleotides (CDN) generated by certain bacteria orby cytosolic dsDNA triggering the activation of a synthase, referred toas cGAS (Cyclic GMP-AMP Synthase, C6orf150, Mab-21 Domain-ContainingProtein).

Given that STING appears to play a pivotal role in controlling a varietyof inflammation driven events, the methods described herein address therole of STING in inflammation aggravated cancer. Using the AOM/DSSmodel, observations similar to MyD88, STING-deficient mice (SKO) aresensitive to CAC suggesting a protective role for STING intumorigenesis. Subsequent analysis indicated that STING signaling andcytokine production was ablated in numerous colon cancer cells analyzed.Data indicates that STING may be a key sensor that promotes theelimination of damaged intestinal epithelial cells. Loss of STINGsignaling may be a common event in colon-associated cancer, an eventthat may enable such cells to escape surveillance from the immunesystem.

Definitions

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

The term “induces or enhances an immune response” is meant causing astatistically measurable induction or increase in an immune responseover a control sample to which a therapeutic has not been administered.Preferably the induction or enhancement of the immune response resultsin a prophylactic or therapeutic response in a subject. Examples ofimmune responses are increased production of type I IFN, increasedresistance to viral and other types of infection by alternate pathogens.The enhancement of immune responses to tumors (anti-tumor responses), orthe development of vaccines to prevent tumors or eliminate existingtumors.

The term “STING” is meant to include, without limitation, nucleic acids,polynucleotides, oligonucleotides, sense and antisense polynucleotidestrands, complementary sequences, peptides, polypeptides, proteins,homologous and/or orthologous STING molecules, isoforms, precursors,mutants, variants, derivatives, splice variants, alleles, differentspecies, and active fragments thereof. STING polynucleotides andpolypeptides are described in U.S. Patent Publications 20130039933 and20110262485.

The term “lacks a functional STING gene” is meant that a transgenicanimal lacks a gene that encodes STING, or lacks other geneticcomponents (e.g. promoters) required for expression of STING.

Unless otherwise indicated, the terms “peptide”, “polypeptide” or“protein” are used interchangeably herein, although typically they referto peptide sequences of varying sizes.

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to a wild typegene. This definition may also include, for example, “allelic,”“splice,” “species,” or “polymorphic” variants. A splice variant mayhave significant identity to a reference molecule, but will generallyhave a greater or lesser number of polynucleotides due to alternatesplicing of exons during mRNA processing. The corresponding polypeptidemay possess additional functional domains or an absence of domains.Species variants are polynucleotide sequences that vary from one speciesto another. Of particular utility in the invention are variants of wildtype gene products. Variants may result from at least one mutation inthe nucleic acid sequence and may result in altered mRNAs or inpolypeptides whose structure or function may or may not be altered. Anygiven natural or recombinant gene may have none, one, or many allelicforms. Common mutational changes that give rise to variants aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

The term “immunoregulatory” is meant a compound, composition orsubstance that is immunogenic (i.e. stimulates or increases an immuneresponse) or immunosuppressive (i.e. reduces or suppresses an immuneresponse).

“An antigen presenting cell” (APC) is a cell that is capable ofactivating T cells, and includes, but is not limited to,monocytes/macrophages, B cells and dendritic cells (DCs). The term“dendritic cell” or “DC” refers to any member of a diverse population ofmorphologically similar cell types found in lymphoid or non-lymphoidtissues. These cells are characterized by their distinctive morphology,high levels of surface MHC-class II expression. DCs can be isolated froma number of tissue sources. DCs have a high capacity for sensitizingMHC-restricted T cells and are very effective at presenting antigens toT cells in situ. The antigens may be self-antigens that are expressedduring T cell development and tolerance, and foreign antigens that arepresent during normal immune processes.

The term “expression vector” as used herein refers to a vectorcontaining a nucleic acid sequence coding for at least part of a geneproduct capable of being transcribed. In some cases, RNA molecules arethen translated into a protein, polypeptide, or peptide. In other cases,these sequences are not translated, for example, in the production ofantisense molecules, siRNA, ribozymes, and the like. Expression vectorscan contain a variety of control sequences, which refer to nucleic acidsequences necessary for the transcription and possibly translation of anoperatively linked coding sequence in a particular host organism. Inaddition to control sequences that govern transcription and translation,vectors and expression vectors may contain nucleic acid sequences thatserve other functions as well.

By “encoding” or “encoded”, “encodes”, with respect to a specifiednucleic acid, is meant comprising the information for translation intothe specified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code.

As used herein, “heterologous” in reference to a nucleic acid is anucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

“Sample” is used herein in its broadest sense. A sample comprisingpolynucleotides, polypeptides, peptides, antibodies and the like maycomprise a bodily fluid; a soluble fraction of a cell preparation, ormedia in which cells were grown; a chromosome, an organelle, or membraneisolated or extracted from a cell; genomic DNA, RNA, or cDNA,polypeptides, or peptides in solution or bound to a substrate; a cell; atissue; a tissue print; a fingerprint, skin or hair; and the like.

The terms “patient”, “subject” or “individual” are used interchangeablyherein, and refers to a mammalian subject to be treated, with humanpatients being preferred. In some cases, the methods of the disclosurefind use in experimental animals, in veterinary application, and in thedevelopment of animal models for disease, including, but not limited to,rodents including mice, rats, and hamsters; primates, cats and dogs.

“Diagnostic” or “diagnosed” means identifying the presence or nature ofa pathologic condition. Diagnostic methods differ in their sensitivityand specificity. The “sensitivity” of a diagnostic assay is thepercentage of diseased individuals who test positive (percent of “truepositives”). Diseased individuals not detected by the assay are “falsenegatives.” Subjects who are not diseased and who test negative in theassay, are termed “true negatives.” The “specificity” of a diagnosticassay is 1 minus the false positive rate, where the “false positive”rate is defined as the proportion of those without the disease who testpositive. While a particular diagnostic method may not provide adefinitive diagnosis of a condition, it suffices if the method providesa positive indication that aids in diagnosis. The terms “treat”,“treated”, “treating” and “treatment”, as used with respect to methodsherein refer to eliminating, reducing, suppressing or ameliorating,either temporarily or permanently, either partially or completely, aclinical symptom, manifestation or progression of an event, disease orcondition. Such treating need not be absolute to be useful. Those inneed of treatment include those already with the disorder as well asthose in which the disorder is to be prevented. In tumor (e.g., cancer)treatment, a therapeutic agent may directly decrease the pathology oftumor cells, or render the tumor cells more susceptible to treatment byother therapeutic agents, e.g., radiation and/or chemotherapy. As usedherein, “ameliorated” or “treatment” refers to a symptom which isapproaches a normalized value (for example a value obtained in a healthypatient or individual), e.g., is less than 50% different from anormalized value, preferably is less than about 25% different from anormalized value, more preferably, is less than 10% different from anormalized value, and still more preferably, is not significantlydifferent from a normalized value as determined using routinestatistical tests. For example the term “treat” or “treating” withrespect to tumor cells refers to stopping the progression of said cells,slowing down growth, inducing regression, or amelioration of symptomsassociated with the presence of said cells. Treatment of an individualsuffering from an infectious disease organism refers to a decrease andelimination of the disease organism from an individual. For example, adecrease of viral particles as measured by plaque forming units or otherautomated diagnostic methods such as ELISA etc.

The “treatment of cancer”, refers to one or more of the followingeffects: (1) inhibition, to some extent, of tumor growth, including, (i)slowing down and (ii) complete growth arrest; (2) reduction in thenumber of tumor cells; (3) maintaining tumor size; (4) reduction intumor size; (5) inhibition, including (i) reduction, (ii) slowing downor (iii) complete prevention, of tumor cell infiltration into peripheralorgans; (6) inhibition, including (i) reduction, (ii) slowing down or(iii) complete prevention, of metastasis; (7) enhancement of antitumorimmune response, which may result in (i) maintaining tumor size, (ii)reducing tumor size, (iii) slowing the growth of a tumor, (iv) reducing,slowing or preventing invasion and/or (8) relief, to some extent, of theseverity or number of one or more symptoms associated with the disorder.

As used herein, the term “safe and effective amount” refers to thequantity of a component which is sufficient to yield a desiredtherapeutic response without undue adverse side effects (such astoxicity, irritation, or allergic response) commensurate with areasonable benefit/risk ratio when used in the manner of this invention.By “therapeutically effective amount” is meant an amount of a compoundof the present invention effective to yield the desired therapeuticresponse. For example, an amount effective to delay the growth of or tocause a cancer, or to shrink the cancer or prevent metastasis. Thespecific safe and effective amount or therapeutically effective amountwill vary with such factors as the particular condition being treated,the physical condition of the patient, the type of mammal or animalbeing treated, the duration of the treatment, the nature of concurrenttherapy (if any), and the specific formulations employed and thestructure of the compounds or its derivatives.

“Cells of the immune system” or “immune cells”, is meant to include anycells of the immune system that may be assayed, including, but notlimited to, B lymphocytes, also called B cells, T lymphocytes, alsocalled T cells, natural killer (NK) cells, natural killer T (NK) cells,lymphokine-activated killer (LAK) cells, monocytes, macrophages,neutrophils, granulocytes, mast cells, platelets, Langerhans cells, stemcells, dendritic cells, peripheral blood mononuclear cells,tumor-infiltrating (TIL) cells, gene modified immune cells includinghybridomas, drug modified immune cells, and derivatives, precursors orprogenitors of the above cell types.

“Immune effector cells” refers to cells capable of binding an antigenand which mediate an immune response selective for the antigen. Thesecells include, but are not limited to, T cells (T lymphocytes), B cells(B lymphocytes), monocytes, macrophages, natural killer (NK) cells andcytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones, andCTLs from tumor, inflammatory, or other infiltrates.

“Immune related molecules” refers to any molecule identified in anyimmune cell, whether in a resting (“non-stimulated”) or activated state,and includes any receptor, ligand, cell surface molecules, nucleic acidmolecules, polypeptides, variants and fragments thereof.

“T cells” or “T lymphocytes” are a subset of lymphocytes originating inthe thymus and having heterodimeric receptors associated with proteinsof the CD3 complex (e.g., a rearranged T cell receptor, theheterodimeric protein on the T cell surfaces responsible for antigen/MHCspecificity of the cells). T cell responses may be detected by assaysfor their effects on other cells (e.g., target cell killing, activationof other immune cells, such as B-cells) or for the cytokines theyproduce.

The phrase “T cell response” means an immunological response involving Tcells. The T cells that are “activated” divide to produce antigenspecific memory T cells or antigen specific cytotoxic T cells. Thecytotoxic T cells bind to and destroy cells recognized as containing theantigen. The memory T cells are activated by the antigen and thusprovide a response to an antigen already encountered. This overallresponse to the antigen is the antigen specific T cell response, e.g.tumor specific.

A “secondary immune response” or “adaptive immune response” may beactive or passive, and may be humoral (antibody based) or cellular thatis established during the life of an animal, is specific for an inducingantigen, and is marked by an enhanced immune response on repeatedencounters with said antigen. A key feature of the T lymphocytes of theadaptive immune system is their ability to detect minute concentrationsof pathogen-derived peptides presented by MHC molecules on the cellsurface.

As used herein, “pharmaceutical composition” refers to a compositionsuitable for administration to a subject animal, including humans andmammals. A pharmaceutical composition comprises a pharmacologicallyeffective amount of a virus or antigenic composition of the inventionand also comprises a pharmaceutically acceptable carrier. Apharmaceutical composition encompasses a composition comprising theactive ingredient(s), and the inert ingredient(s) that make up thepharmaceutically acceptable carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients. Accordingly, thepharmaceutical compositions of the present invention encompass anycomposition made by admixing a compound or conjugate of the presentinvention and a pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” include any andall clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, buffers, and excipients, such as a phosphate buffered salinesolution, 5% aqueous solution of dextrose or mannitol, and emulsions,such as an oil/water or water/oil emulsion, and various types of wettingagents and/or adjuvants. Suitable pharmaceutical carriers andformulations are described in Remington's Pharmaceutical Sciences, 19thEd. (Mack Publishing Co., Easton, 1995). Pharmaceutical carriers usefulfor the composition depend upon the intended mode of administration ofthe active agent. Typical modes of administration include, but are notlimited to, enteral (e.g., oral) or parenteral (e.g., subcutaneous,intramuscular, intravenous or intraperitoneal injection; or topical,transdermal, or transmucosal administration). A “pharmaceuticallyacceptable salt” is a salt that can be formulated into a compound orconjugate for pharmaceutical use including, e.g., metal salts (sodium,potassium, magnesium, calcium, etc.) and salts of ammonia or organicamines.

As used herein, “pharmaceutically acceptable” or “pharmacologicallyacceptable” refers to a material which is not biologically or otherwiseundesirable, i.e., the material may be administered to an individualwithout causing any undesirable biological effects or interacting in adeleterious manner with any of the components of the composition inwhich it is contained, or when administered using routes well-known inthe art, as described below.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample.

Labels

In some embodiments, the STING, cGAS or other molecule is labeled tofacilitate its detection. A “label” or a “detectable moiety” is acomposition detectable by spectroscopic, photochemical, biochemical,immunochemical, chemical, or other physical means. For example, labelssuitable for use in the present invention include, but are not limitedto, radioactive labels (e.g., ³²P), fluorophores (e.g., fluorescein),electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin, digoxigenin, or haptens as well as proteins which can be madedetectable, e.g., by incorporating a radiolabel into the hapten orpeptide, or used to detect antibodies specifically reactive with thehapten or peptide.

Examples of labels suitable for use in the present invention include,but are not limited to, fluorescent dyes (e.g., fluoresceinisothiocyanate, Texas red, rhodamine, and the like), radiolabels (e.g.,3H ¹²⁵I, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase,alkaline phosphatase and others commonly used in an ELISA), andcolorimetric labels such as colloidal gold, colored glass or plasticbeads (e.g., polystyrene, polypropylene, latex, etc.).

The label may be coupled directly or indirectly to the desired componentaccording to methods well known in the art. Preferably, the label in oneembodiment is covalently bound to the molecule using an isocyanatereagent for conjugation of an active agent according to the invention.In one aspect of the invention, the bifunctional isocyanate reagents ofthe invention can be used to conjugate a label to a target molecule toform a label target molecule conjugate without an active agent attachedthereto. The label target molecule conjugate may be used as anintermediate for the synthesis of a labeled conjugate according to theinvention or may be used to detect the target molecule conjugate. Asindicated above, a wide variety of labels can be used, with the choiceof label depending on sensitivity required, ease of conjugation with thedesired component, stability requirements, available instrumentation,and disposal provisions. Non-radioactive labels are often attached byindirect means. Generally, a ligand molecule (e.g., biotin) iscovalently bound to the target molecule. The ligand then binds toanother molecule (e.g., streptavidin), which is either inherentlydetectable or covalently bound to a signal system, such as a detectableenzyme, a fluorescent compound, or a chemiluminescent compound.

The STING, cGAS or other molecule contemplated herein for use in themethods can also be conjugated directly to signal-generating compounds,e.g., by conjugation with an enzyme or fluorophore. Enzymes suitable foruse as labels include, but are not limited to, hydrolases, particularlyphosphatases, esterases and glycosidases, or oxidotases, particularlyperoxidases. Fluorescent compounds, i.e., fluorophores, suitable for useas labels include, but are not limited to, fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Further examples of suitable fluorophores include, but are not limitedto, eosin, TRITC-amine, quinine, fluorescein W, acridine yellow,lissamine rhodamine, B sulfonyl chloride erythroscein, ruthenium (tris,bipyridinium), Texas Red, nicotinamide adenine dinucleotide, flavinadenine dinucleotide, etc. Chemiluminescent compounds suitable for useas labels include, but are not limited to, luciferin and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems that can be used in the methods ofthe present invention, see U.S. Pat. No. 4,391,904.

Means for detecting labels are well known to those of skill in the art.Thus, for example, where the label is radioactive, means for detectioninclude a scintillation counter or photographic film, as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence. The fluorescence may bedetected visually, by the use of electronic detectors such as chargecoupled devices (CCDs) or photomultipliers and the like. Similarly,enzymatic labels may be detected by providing the appropriate substratesfor the enzyme and detecting the resulting reaction product.Colorimetric or chemiluminescent labels may be detected simply byobserving the color associated with the label. Other labeling anddetection systems suitable for use in the methods of the presentinvention will be readily apparent to those of skill in the art. Suchlabeled modulators and ligands can be used in the diagnosis of a diseaseor health condition.

Formulation of Pharmaceutical Compositions

To administer compositions of the present disclosure to human or testanimals, it is preferable to formulate the active agent in a compositioncomprising one or more pharmaceutically acceptable carriers. The phrase“pharmaceutically or pharmacologically acceptable” refer to molecularentities and compositions that do not produce allergic, or other adversereactions when administered using routes well-known in the art, asdescribed below. “Pharmaceutically acceptable carriers” include any andall clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The compositions are administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic. Other administrationmethods are contemplated, including topical, particularly transdermal,transmucosal, rectal, oral or local administration, e.g. through acatheter placed close to the desired site.

Pharmaceutical compositions of the present disclosure containing theactive agent described herein may contain pharmaceutically acceptablecarriers or additives depending on the route of administration. Examplesof such carriers or additives include water, a pharmaceutical acceptableorganic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, acarboxyvinyl polymer, carboxymethylcellulose sodium, polyacrylic sodium,sodium alginate, water-soluble dextran, carboxymethyl starch sodium,pectin, methyl cellulose, ethyl cellulose, xanthan gum, gum Arabic,casein, gelatin, agar, diglycerin, glycerin, propylene glycol,polyethylene glycol, Vaseline, paraffin, stearyl alcohol, stearic acid,human serum albumin (HSA), mannitol, sorbitol, lactose, apharmaceutically acceptable surfactant and the like. Additives used arechosen from, but not limited to, the above or combinations thereof, asappropriate, depending on the dosage form of the present disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the composition to be administeredcan be prepared in a physiologically acceptable vehicle or carrier. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. Intravenous vehicles can includevarious additives, preservatives, or fluid, nutrient or electrolytereplenishers.

A variety of aqueous carriers, e.g., sterile phosphate buffered salinesolutions, bacteriostatic water, water, buffered water, 0.4% saline,0.3% glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Therapeutic formulations are prepared for storage by mixing the activeagent having the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Aqueous suspensions may contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan mono oleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

The active agents described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of active agent in these formulations can vary widely,for example from less than about 0.5%, usually at or at least about 1%to as much as 15 or 20% by weight and will be selected primarily basedon fluid volumes, viscosities, etc., in accordance with the particularmode of administration selected. Thus, a typical pharmaceuticalcomposition for parenteral injection could be made up to contain 1 mlsterile buffered water, and 50 mg of active agent. A typical compositionfor intravenous infusion could be made up to contain 250 ml of sterileRinger's solution, and 150 mg of antibody. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed., Mack PublishingCompany, Easton, Pa. (1980). An effective dosage of active agent iswithin the range of 0.01 mg to 1000 mg per kg of body weight peradministration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the present disclosure are wellbalanced, thereby enhancing their utility for both in vitro andespecially in vivo uses, while other compositions lacking such balanceare of substantially less utility. Specifically, compositionscontemplated for use in the disclosure have an appropriate degree ofsolubility in aqueous media which permits absorption and bioavailabilityin the body, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membrane to a putative siteof action. Thus, antibody compositions contemplated are maximallyeffective when they can be delivered to the site of target antigenactivity.

Administration and Dosing

In one aspect, methods of the present disclosure include a step ofadministering a pharmaceutical composition. In certain embodiments, thepharmaceutical composition is a sterile composition.

Methods of the present disclosure are performed using anymedically-accepted means for introducing therapeutics directly orindirectly into a mammalian subject, including but not limited toinjections, oral ingestion, intranasal, topical, transdermal,parenteral, inhalation spray, vaginal, or rectal administration. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intracisternal injections, as well as catheter orinfusion techniques. Administration by, intradermal, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection andor surgical implantation at a particular site is contemplated as well.

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the disclosure implanted near or at site of the cancer.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, every other day, twice weekly, three timesweekly, weekly, every 2 weeks, every 3 weeks, monthly, or at a longerinterval.

Also contemplated in the present disclosure is the administration ofmultiple agents, such as the active agent compositions in conjunctionwith a second agent as described herein, including but not limited to achemotherapeutic agent. Suitable chemotherapeutic agents include:daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al.,(1986) supra) and those listed in the Table below.

TABLE I Alkylating agents Nitrogen mustards mechlorethaminecyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureascarmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)Ethylenimine/Methyl-melamine thriethylenemelamine (TEM) triethylenethiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine) Alkylsulfonates busulfan Triazines dacarbazine (DTIC) Antimetabolites FolicAcid analogs methotrexate Trimetrexate Pemetrexed (Multi-targetedantifolate) Pyrimidine analogs 5-fluorouracil fluorodeoxyuridinegemcitabine cytosine arabinoside (AraC, cytarabine) 5-azacytidine2,2⁻-difluorodeoxy-cytidine Purine analogs 6-mercaptopurine6-thioguanine azathioprine 2′-deoxycoformycin (pentostatin)erythrohydroxynonyl-adenine (EHNA) fludarabine phosphate2-chlorodeoxyadenosine (cladribine, 2-CdA) Type I TopoisomeraseInhibitors camptothecin topotecan irinotecan Biological responsemodifiers G-CSF GM-CSF Differentiation Agents retinoic acid derivativesHormones and antagonists Adrenocorticosteroids/antagonists prednisoneand equiv-alents dexamethasone ainoglutethimide Progestinshydroxyprogesterone caproate medroxyprogesterone acetate megestrolacetate Estrogens diethylstilbestrol ethynyl estradiol/equivalentsAntiestrogen tamoxifen Androgens testosterone propionatefluoxymesterone/equivalents Antiandrogens flutamidegonadotropin-releasing hormone analogs leuprolide Nonsteroidalantiandrosens flutamide Natural products Antimitotic drugs Taxanespaclitaxel Vinca alkaloids vinblastine (VLB) vincristine vinorelbineTaxotere ® (docetaxel) estramustine estramustine phosphateEpipodophylotoxins etoposide teniposide Antibiotics actimomycin Ddaunomycin (rubido-mycin) doxorubicin (adria-mycin)mitoxantroneidarubicin bleomycin splicamycin (mithramycin) mitomycinCdactinomycin aphidicolin Enzymes L-asparaginase L-arginaseRadiosensitizers metronidazole misonidazole desmethylmisonidazolepimonidazole etanidazole nimorazole RSU 1069 E09 RB 6145 SR4233nicotinamide 5-bromodeozyuridine 5-iododeoxyuridine bromodeoxycytidineMiscellaneous agents Platinium coordination complexes cisplatinCarboplatin oxaliplatin Anthracenedione mitoxantrone Substituted ureahydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)procarbazine Adrenocortical suppressant mitotane (o,p⁻- DDD)ainoglutethimide Cytokines interferon (a, 3, y) interleukin-2Photosensitizers hematoporphyrin derivatives Photofrin ® benzoporphyrinderivatives Npe6 tin etioporphyrin (SnET2) pheoboride-abacteriochlorophyll-a naphthalocyanines phthalocyanines zincphthalocyanines Radiation X-ray ultraviolet light gamma radiationvisible light infrared radiation microwave radiation

The amounts of active agent composition in a given dosage may varyaccording to the size of the individual to whom the therapy is beingadministered as well as the characteristics of the disorder beingtreated. In exemplary treatments, it may be necessary to administerabout 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day,100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000mg/day. These concentrations may be administered as a single dosage formor as multiple doses. Standard dose-response studies, first in animalmodels and then in clinical testing, reveals optimal dosages forparticular disease states and patient populations.

Also contemplated in the present disclosure, the amounts of active agentin a given dosage may vary according to the size of the individual towhom the therapy is being administered as well as the characteristics ofthe disorder being treated. It will also be apparent that dosing may bemodified if traditional therapeutics are administered in combinationwith therapeutics of the disclosure.

Exemplary conditions or disorders that can be treated using the presentmethods include cancers, such as esophageal cancer, pancreatic cancer,metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas,bladder cancer, stomach cancer, fibrotic cancer, glioma, malignantglioma, diffuse intrinsic pontine glioma, recurrent childhood brainneoplasm renal cell carcinoma, clear-cell metastatic renal cellcarcinoma, kidney cancer, prostate cancer, metastatic castrationresistant prostate cancer, stage IV prostate cancer, metastaticmelanoma, melanoma, malignant melanoma, recurrent melanoma of the skin,melanoma brain metastases, stage MA skin melanoma; stage MB skinmelanoma, stage IIIC skin melanoma; stage IV skin melanoma, malignantmelanoma of head and neck, lung cancer, non small cell lung cancer(NSCLC), squamous cell non-small cell lung cancer, breast cancer,recurrent metastatic breast cancer, hepatocellular carcinoma, hodgkin'slymphoma, follicular lymphoma, non-hodgkin's lymphoma, advanced B-cellNHL, HL including diffuse large B-cell lymphoma (DLBCL), multiplemyeloma, chronic myeloid leukemia, adult acute myeloid leukemia inremission; adult acute myeloid leukemia with Inv(16)(p13.1q22);CBFB-MYH11; adult acute myeloid leukemia with t(16;16)(p13.1;q22);CBFB-MYH11; adult acute myeloid leukemia with t(8;21)(q22;q22);RUNX1-RUNX1T1; adult acute myeloid leukemia with t(9;11)(p22;q23);MLLT3-MLL; adult acute promyelocytic leukemia with t(15;17)(q22;q12);PML-RARA; alkylating agent-related acute myeloid leukemia, chroniclymphocytic leukemia, richter's syndrome; waldenstrom macroglobulinemia,adult glioblastoma; adult gliosarcoma, recurrent glioblastoma, recurrentchildhood rhabdomyosarcoma, recurrent ewing sarcoma/peripheral primitiveneuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma,colorectal cancer, MSI positive colorectal cancer; MSI negativecolorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrentnasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma;cervical adenosquamous carcinoma; cervical squamous cell carcinoma;recurrent cervical carcinoma; stage IVA cervical cancer; stage IVBcervical cancer, anal canal squamous cell carcinoma; metastatic analcanal carcinoma; recurrent anal canal carcinoma, recurrent head and neckcancer; carcinoma, squamous cell of head and neck, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer,gastric cancer, advanced GI cancer, gastric adenocarcinoma;gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissuesarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrentmerkel cell carcinoma; stage III merkel cell carcinoma; stage IV merkelcell carcinoma, and myelodysplastic syndrome.

Example 1

Activation of STING-Dependent Genes by AOM.

Given that chronic inflammation is known to aggravate colon cancer andthat STING has been shown to influence inflammatory responses,especially that invoked by cytosolic self or pathogen related DNA, therole of STING in the control of inflammatory colitis-associatedcarcinogenesis (CAC) was examined. Towards these objectives, the AOM/DSSmodel, as described above, was utilized and analyzed the effects of AOMand precursor DMH upon STING signaling. Principally, wild type (WT) orSTING-deficient (SKO) murine embryonic fibroblasts (MEF) were treated invitro with DMH or metabolite AOM for 8 hours and microarray analysisperformed to analyze the consequences to gene expression. This studyindicated that AOM activated mRNA production of a wide array of innateimmune related genes in WT cells, including IFIT3 and Cxcl2 (FIG. 1A;FIG. 7). However, there was a marked decrease in the production of thesame genes in cells lacking STING (SKO) indicating that AOM was indeedcapable of activating the STING pathway (FIG. 1A, left panel). A similareffect was observed following the treatment of cells with DMH (FIG. 1A,right panel). The observed STING-dependent gene expression was confirmedfollowing RT-PCR analysis of select mRNA such as Cxcl10 and IFIT3 (FIG.1B). Similarly normal human colon epithelial cells (FHC) were treatedwith AOM and a similar induction of innate immune genes was observed,controlled by STING, including Cxcl10 (FIG. 1C). The production ofCxcl10 by AOM was similarly reduced in FHC's treated with RNAi to STING(FIG. 1D). Thus, the DNA damaging agent DMH/AOM can invokeSTING-dependent signaling.

To start determining the mechanisms underlining the cause ofDMH/AOM-induced STING activity, MEF or FHC cells were treated with thesedrugs and observed by two different approaches, DAPI staining andimmunofluorescence (IF) using anti-dsDNA antibody, the leakage of DNAinto the cytosol (FIGS. 8A and 8B). Cytosolic DNA activates STING andstimulates STING/TBK1 trafficking via autophagy to endosomal regionsharboring the transcription factors IRF3 and NF-κB, which triggerscytokine production. Thus, to determine the consequences of DMH/AOMtreatment upon STING's ability to activate these key transcriptionfactors, IF analysis on FHC'S treated with these drugs was carried out.This study indicated that DMH/AOM could instigate the translocation ofIRF3/NF-κKB in treated cells (FIG. 8C). Thus, DMH/AOM inducesSTING-dependent signaling conceivably through the leaking of DNA intothe cytyosol (FIG. 1A; FIGS. 8A and 8B).

Loss of STING renders mice susceptible to CAC.⋅ The data indicates thatDMH/AOM can activate STING in vitro. To examine the consequences of thisin vivo, mice were treated once with AOM and subsequently orally with 4treatments of DSS. Prior to this, STING expression in the intestine wasanalyzed by IHC. This study showed that STING was expressed in laminapropria cells as well as in endothelial and epithelial cells of thegastrointestinal tract (FIG. 1E). After 13 weeks the mice were analyzedfor tumor development in the colon. Surprisingly, the mice lacking STING(SKO) developed colonic tumors at a much higher frequency compared towild type mice (WT) (FIG. 2A-C; FIG. 9A). Indeed, 4/7 WT mice exhibitedtumor formation compared to 7/7 SKO within the same time period (FIGS.2A and 2B; FIG. 9A), Hematoxylin and eosin (HE) analysis confirmed thatAOM/DSS treated SKO mice exhibited significant inflammatory cellinfiltration and development of adenocarcinoma in colon, compared to WTmice (FIG. 2B; FIG. 9B). However, microarray analysis indicated thattumors from WT mice exhibited higher levels of select gene expression,such as Cxcl13 and Ccr6, compared to tumors retrieved from SKO mice,perhaps since loss of STING suppressed immunomodulatory transcriptionalevents (FIGS. 2E and 2F). It is postulated that STING may recognizedamaged DNA and activate the production of cytokines that conceivablycould promote tissue repair or stimulate the immune system to eradicatesuch cells. Thus, loss of STING may enable damaged cells to escapeimmune surveillance processes and progress more readily into tumors.

Chronic STING activation is responsible for inflammatory bowel disease.Transient STING activation in response to cell damaging agents such asAOM and dextran sulphate (DSS) facilitates wound healing. Thus, loss ofSTING may lead to a lack of colonic repair and the infiltration ofgenotoxic microbiota that aggravate STING-independent inflammatoryresponses. However, chronic irritation of STING in wild type mice byagents such as DSS can lead to inflammatory bowel disease (IBD). Thus,suppression of STING activity by inhibitors/drugs/compounds may lead toa reduction in IBD such as Crohn's disease and ulcerative colitis.

Suppression of IL22BP expression in STING-deficient mice. Indemonstrating that loss of STING facilitated colon cancer development,the tumor suppressive mechanisms associated with STING activity remainto be clarified. It remains plausible that STING could exert directtumor suppressive, growth inhibitory or pro-apoptotic properties similarto tumor suppressor p53. Further, AOM treatment has been known to inducefrequent Ras mutations, which in the context of loss of STING, couldfacilitate cellular transformation. Expression of oncogenic Ras in anenvironment where p53 function is lost renders normal cells the abilityto form foci in soft agar and to become tumorigenic. To evaluate thispossibility, WT, SKO, or p53-deficient MEFs were transfected as positivecontrols, with Myc or activated Ras and cellular transformationmonitored. MEF's lacking p53 were found to be readily transformed by theintroduction of Myc or activated Ras. However, MEF's lacking STING didnot appear appreciably sensitive to transformation by overexpression ofMyc, or activated Ras. Thus, the absence of STING does not appear toexert an oncogenic stimulus, at least in vitro or to cooperate with Mycor Ras in the cellular transformation process.

However, it has been demonstrated that mice lacking certain cytokinessuch as IL-18, IL-22 or the innate immune adaptor MyD88 are similarlysusceptible to AOM/DSS induced CAC. In this situation, MyD88 exerts aprotective effect by facilitating the production of IL-18 through theIL-18R, which is required to inhibit IL-22BP. IL-22BP is necessary tosuppress IL-22 function, which can promote the proliferation ofintestinal epithelial cells following damage by carcinogens orinflammatory agents. Mice lacking IL-18 or IL-22BP are highlysusceptible to CAC, similar to STING-deficient mice. It was noted fromthe microarray analyses that IL-18 levels were reduced in SKO MEFstreated with STING-activating dsDNA (dsDNA90 base pairs) (FIG. 3A).Therefore investigation of the involvement of STING on the possibleregulation of the IL-18/IL-22BP axis was made. First, a confirmation ofthe influence of STING upon IL-18 expression was made since it wasadditionally noted that the promoter of this cytokine is known to harbornumerous sites recognized by innate immune gene activating transcriptionfactors such as STAT1, NF-κB, IRF1 and IRF7. The analysis indicated thatIL-18, which is expressed in a wide variety of cell types, is a STINGinducible gene, as determined following treatment of MEF cells withdsDNA or STING-activating CDN's (cGAMP) (FIG. 3A). A similar studyindicated that DMH/AOM could also trigger the production of IL-18 indendritic cells, in a STING-dependent manner, confirming that IL-18 canindeed be induced through the STING pathway.

Following the confirmation, an examination of whether DSS treatmentcould affect IL-18 and IL-22BP expression in vivo was made. Following 7days of DSS treatment, colons were retrieved from WT or SKO mice andIL-18, IL-22BP or IL-22 expression analyzed by RT-PCR. This studyindicated that IL-18 expression was reduced in mice lacking STING (SKO)after 2 days treatment (FIG. 3B). However, a much more pronounceddecrease in IL-22BP expression, a protein predominantly expressed fromCD1 1c⁺ dendritic cells, was observed in SKO mice compared to WT mice(FIG. 3B). The expression of IL-22, in contrast, remained relativelyunaffected. It was surprising to note that IL-22BP levels weresuppressed in the absence of STING, where IL-18 levels were noted to bealso relatively low. However, it has been reported that downregulationof IL22-BP can occur even in the absence of IL-18, indicating that otherSTING-dependent factors may also contribute to the regulation ofIL-22BP. To complement this study, control or SKO mice with AOM/DSSregimes were treated and after 13 weeks again analyzed IL-22BPexpression levels in normal or tumor tissue taken from the treated mice.Analogous to DSS treatment alone, observations noted the greatly reducedexpression of IL-22BP in the tumors of SKO mice compared to WT mice(FIG. 3C). However, IL-18 and IL-22 levels appeared less dramaticallyaffected. Taken together, it is conceivable that DNA damage or thesensing of microbial ligands that may invade colon tissue afterintestinal damage (for example by DSS) can trigger STING activityleading to the production of IL-18. This event would suppress IL-22BPproduction and enable IL-22 to facilitate tissue repair. However, itappears that loss of STING function in the long term also influencesIL-22BP production which is critical for controlling the growthstimulator properties of IL-22. This event may be mediated by microbestriggering STING-dependent innate immune pathways that control IL-22BPproduction. Data thus indicates that similar to mice lacking MyD88,IL-18 or IL-22BP, STING-deficient mice are also prone to CAC induced byAOM/DSS.

STING activity is suppressed in colonic tumor cells. Data indicates thatSTING is required to protect against carcinogens and perhaps microbesthat facilitate inflammation driven CAC. STING conceivably senses DNAdamage and signals the event to anti-tumor immunosurveillance cells.Dendritic cells (DC) such as CD8 alpha DCs engulf tumor cells ornecrotic tumor cell debris, and the DNA from the engulfed cell or debrisactivates STING extrinsically in the DC. This leads to the production ofcytokines that are essential for antitumor T cell responses. Given this,STING signaling in human colonic cells was analyzed, since it wasfeasible that defects in STING function may enable damaged cells toevade the immune system. To thus evaluate STING signaling in humancolonic cancer cells, STING expression in hTERT and (FHC) intestinalepithelial cells as controls were examined. STING was found to beexpressed in these cells and to produce type I IFN in response totransfected cytosolic DNA (dsDNA 90 base pairs), which is known to be aSTING-dependent event (FIG. 4A-C). A similar inducible effect by dsDNAwas noted following measurement by Cxcl10, also a highlydsDNA-inducible, STING regulated gene (FIG. 4D). Next, examination ofthe expression of STING in a variety of tumor cells isolated fromvarious stages of colon cancer was done and it was found that 10/11 celllines expressed STING (FIG. 4A; FIG. 12). However, the STING pathway wasdefective in the majority of cells analyzed (>80%) and such cells couldnot efficiently produce type I IFN in response to cytosolic dsDNA (FIGS.4A-C). Only two out the 11 cell lines analyzed (SW116 and LS123)appeared only somewhat able to produce type I IFN in response tocytosolic DNA stimulation. In contrast, transfected synthetic dsRNA(polyL⋅C) was able to stimulate the production of type I IFN relativelywell in all but 3 of the cancer cells, likely indicating that theRIGI/MDA5 pathway was functional. To confirm this analyses DNAmicroarray analyses on the colon cancer cells following stimulation ofthe STING pathway using transfected cytosolic DNA was carried out. Thisdata indicated significant suppression of STING-dependent primary innateimmune gene activation in the colon cancer cells analyzed, includingtype I IFN, as well as a host of other key innate immune modulatorygenes such as CXCL10, CXCL11 and CC15 (FIGS. 4E and 4F). LoVo cells wereobserved to retain some ability to induce cytosolic DNA-induced cells,while HT116 cells appeared significantly defective in STING-dependentsignaling. The data indicates that STING-dependent innate immunepathways appear to be preferentially deregulated in colonic tumor lines.

To analyze the mechanisms of STING inactivation further,immunohistochemical (MC) analyses were carried out on the normal or thecolon tumor cells. It had been previously shown that in the presence ofcytosolic DNA, STING becomes activated and translocates via anon-canonical autophagy associated process required for the activationof transcription factors NF-κB and IRF3 (interferon regulatory factor3). Thus, blockage of STING translocation through use of Brefeldin A orthrough suppression of the key autophagy modulator VPS34 inhibits STINGsignaling function. Thus, normal or human colon cancer cells weretransfected with dsDNA to activate STING and it was observed that only4/11 cell-lines (LS123, SW480, LoVo and HT29) exhibited evidence ofSTING trafficking (FIG. 5A). This study was then completed by analyzingthe translocation of the transcription factors NF-κB and IRF3 insimilarly treated cells using IHC techniques. It was observed that IRF3was able to translocate in cells that facilitated STING trafficking,except for SW480 (FIG. 5B). This may explain, in part, the partialstimulation of innate immune genes in some of the cells analyzed bymicroarray, such as LoVo. However, only two of the cells (LS1116, LS123)exhibited translocation of the NF-κB subunit p65 (FIG. 5C). Since bothIRF3 and NF-κB are required for optimal transcription of type I IFN, itmay explain why LS1116 and LS123 remained partially able to stimulatetype I IFN production, following treatment with cytosolic DNA, while theremainder did not (FIGS. 4B and 4C). However, cytosolic DNA-dependent,STING-controlled signaling remained severely defective in all coloncancer types examined as shown (FIG. 4).

To extend these findings further, immunoblot analyses were carried outon the normal or colon tumor cells. In the presence of cytosolic DNA,STING undergoes phosphorylation and is then degraded, an event thatfacilitates its activity, perhaps through releasing TBK1 tophosphorylate IRF3. First, STING phosphorylation/degradation afteractivation was impeded in many cell lines analyzed, which may affectSTING function (for example, LS123, SW480, SW1417, HT116) (FIG. 5D).Confirmation was made in tumor cells that exhibited some STINGtrafficking (LS1116, LS123, LoVo and HT29) that the IRF3 kinaseactivator Tank binding kinase 1 (TBK1) underwent phosphorylation in thepresence of cytosolic DNA (FIG. 5D). Accordingly, observations ofphospho-IRF3 activity in cells with active TBK1 (FIG. 5D) were made. Theremainder of the tumor cells, such as SW480, SW1417, SW48 and HT116 didnot exhibit phospho-TBK1 activity or IRF3 translocation, likely due toan inability of STING to undergo autophagy, or since STING expressionwas completely absent as in the case of SW48 (FIG. 5D). Surprisingly,observations showed that the vast majority of cells lacked p65translocation, phosphorylation of p65 was evident (for example LoVo,HT29, SW480 and SW1417). This may suggest that NF-κB signaling could bedefective at the level of p65 translocation. Taken together the studyclearly indicates that STING-signaling is defective in a wide variety ofcolon cancer cells examined.

Cyclic dinucleotides (CDN's) have been shown to activate STING. CDN'shave been shown to be generated through cytosolic dsDNA speciestriggering the activation of a synthase, referred to as cGAS (CyclicGMP-AMP Synthase, C6orf150, Mab-21 Domain-Containing Protein). Loss ofcGAS has thus been shown to affect STING signaling. To complement theabove study, the expression of cGAS in the colon cancer cell-lines wasexamined. This analysis indicated that 5/11 colon cancer cells hadundetectable levels of cGAS expression, an event that correlated withloss of STING translocation and TBK1/IRF3 activity (FIGS. 5D and 5Ehighlighted by dashed line or box). Interestingly, loss of cGASexpression could be rescued using de-methylating agents lines (SW480,HT116) indicating that some cells exhibited suppressed cGAS promoteractivity (FIG. 5F). However, rescue of cGAS expression did not robustlyrescue STING activity as determined (FIGS. 13A and 13B) indicating thatfurther defects in STING signaling may exist in these cells. Given thesefindings, examination of the expression of cGAS in 47 human coloncancers at various stages of tumorigenesis was analyzed (FIG. 13C).Expression of cGAS was low to undetectable in approximately 30% of cellsanalyzed. Thus, defects in cGAS or STING expression, or signaling appeardefective in a large number of tumor colon cancer cells lines and couldconstitute a major cause of tumorigenesis. It should be noted, however,that similarly defective STING-signaling was found in a wide variety ofother tumor cells examined, indicating that defects in STING functioncould be common in cells other than those of the colon (FIG. 14).

Cancer cells with Defective STING Signaling are susceptible to viraloncolysis. Numerous cancer cells have been shown to be defective inantiviral responses, although the mechanisms remain to be fullydetermined. Indeed, a variety of viruses are now being used in theclinic to determine their efficacy as anti-tumor therapeutics, includingherpes simplex 1 (HSV1) which harbors a dsDNA genome. HSV1 has beenshown to potently trigger innate immune responses through activating theSTING. Mice deficient in STING signaling are extremely sensitive tolethal HSV1 infection since they lack the ability to mount anappropriate innate immune response, including the generation of type IIFN. Given the findings that STING signaling is defective in a largenumber of cancer cells, their susceptibility to HSV1 infection wasexamined. First, analysis of the response to a recombinant HSV1 thatexpresses luciferase (HSV1-Luc) was made. This analysis indicated thatcolon cancer cells exhibiting defective STING signaling enabled highlevels of HSV1-Luc expression (FIG. 6A). However, the two cancer lines(SW116 and LS123), which exhibited partial STING-dependent innate immuneresponses (FIG. 4B-D), as well as the control hTERT and FHC's did notfacilitate robust HSV-Luc gene expression (FIG. 6A). This coincided withthe normal cells and SW116 and LS123 responding to infection byproducing CXCL10, similar to their response to dsDNA (FIGS. 6B and 6C;FIG. 4C). None of the tumor cells harboring severely defective STINGfunction could robustly produce type I IFN after infection. To extendthis study, an HSV construct that lacked the γ34.5 gene (HSV1γ34.5) wasused, encoding viral protein that has been reported to inhibit hostdefense in part through preventing host cell translational shut-off. Asimilar virus that lacks γ34.5 is being examined in the clinic as ananticancer agent. It was observed that colon cancer cells defective inSTING-signaling were unable to mount an efficient type I IFN responsefollowing infection with HSV1γ34.5 (FIG. 6D). Thus, the examination ofSTING-signaling may be a useful prognostic marker for whether HSV1 orother viral based anti-cancer therapies will be efficacious for thetreatment of malignant disease.

Experimental Procedure

Mice: STING knockout mice (SKO, Sting^(i-)) were generated in theUniversity of Miami laboratory (Ichikawa 2008). Wild type mice (WT) wereused as control groups. Mice care and study were conducted underapproval from the Institutional Animal Care and Use Committee (IACUC) ofthe University of Miami.

Acute DSS colitis. WT and SKO mice 6-8 weeks of ages were divided intoexperimental and control groups. Mice in experimental group received 3%Dextran sodium sulfate (DSS, MP 160110; MW 36000-5000) for 5 days,followed by 2 days of regular drinking water. Distilled water wasadministered into control group mice.

AOM/DSS Induced Colitis-Associated Tumor Induction: WT and SKO mice wereinjected intraperitoneally with Azoxymethane (AOM; MP 180139; MW 74.08)at a dose of 10 mg/kg. DSS at 5% which was administered in the drinkingwater for 7 days every 3 weeks. DSS cycle was repeated 4 times. On 91days, micro endoscopic procedure was performed in a blinded fashion forcounting number of polyps. Mice were sacrificed at day 121 and colon wasresected, flushed with PBS, fixed in formalin for histology and frozenfor RNA expression analysis.

Primary cell culture: Mouse embryonic fibroblasts (MEFs) were obtainedfrom e15 embryos by a standard procedure as described. Bone marrowderived dendritic cells were isolated from hind-limb femurs of 8-10weeks old mice. Briefly, the marrow cells were flushed from the boneswith Dulbecco's modified eagle medium (DMEM, Invitrogen), 10%heat-inactivated fetal calf serum (FCS, Invitrogen) with a 23 gaugeneedle and incubated at 37° C. for 4 hours. After harvesting floatingcells, approximately 2×10⁷ cells were seeded in 10 cm dish with completeDMEM including 10 ng/ml of Recombinant mouse GM-CSF (GM-CSF, BioLegend)for CD11c positive dendritic cells. After 1 week, bone marrow deriveddendritic cells were obtained. Normal human colon epithelial cells andcolon cancer cell lines were purchased from ATCC and cultured in theirappropriate growth media according to the ATCC instructions. Media andsupplements are from Invitrogen. hTERT-BJ1 Telomerase Fibroblasts(hTERT) were originally purchased from Clontech and were cultured in 4:1ratio of DMEM:Medium 199 supplement with 10% FBS, 4 mM L-Glutamine and 1mM sodium pyruvate at 37° C. in a 5% CO2-humidified atmosphere.

Gene array analysis: Total RNA was isolated from cells or tissues withRNeasy Mini kit (74104, Qiagen, Valencia, Calif.). RNA quality wasanalyzed by Bionalyzer RNA 6000 Nano (Agilent Technologies, Santa ClaraCalif.). Gene array analysis was examined by Illumina Sentrix BeadChipArray (Mouse WG6 version 2) (Affymetrix, Santa Clara Calif.) at theOncogenomics Core Facility, University of Miami. Raw intensity valuesfrom Illumina array are uploaded on GeneSpring™ software from Agilent.Values are Quantile normalized and log 2 transformed to the median ofall samples. Significantly differential expressed genes are computedusing the Student's t-test and selected using threshold of P-value<0.05. Hierarchical Clustering and visualization of selecteddifferentially expressed genes is performed on GeneSpring using PearsonCorrelation distance method and linkage was computed using the Wardmethod. Gene expression profiles were processed and statistical analysiswas performed at the Sylvester Comprehensive Cancer CenterBioinformatics Core Facility University of Miami.

Histopathology. Mice were sacrificed and the colon tissues were fixed in10% formalin for 48 hours. All processes for paraffin block andHematoxylin and Eosin staining (H&E) were performed at the pathologyresearch resources histology laboratory in University of Miami.

Statistical Analysis: All statistical analysis was performed byStudent's t test unless specified. The data was considered to besignificantly different when P<0.05.

Supplemental Information

Quantitative Real time PCR (qPCR): Total RNA were reverse-transcribedusing M-MLV Reverse Transcriptase (Promega). Real-time PCR was performedusing Taqman Gene Expression Assay (Applied Biosystems) for innateimmune genes and inflammatory cytokines (Cxc//0: Mm00445235, Ifit3:Mm0170846).

Immunoblot analysis: Equal amounts of proteins were resolved on sodiumdodecyl sulfate (SDS)—Polyacrylamide gels and then transferred topolyvinylidene fluoride (PVDF) membranes (Millipore). After blockingwith 5% Blocking Reagent, membranes were incubated with various primaryantibodies (and appropriate secondary antibodies). The image wasresolved using an enhanced chemiluminescence system ECL (ThermoScientific) and detected by autoradiography. Antibodies: rabbitpoyclonal antibody against STING was developed in the laboratory; otherantibodies were obtained from following sources: β-actin (SigmaAldrich), p-IRF3 (Cell Signaling), IRF3 (Santa Cruz Biotechnology), p-TBK1, TBK1, p-p65, p65.

Interferon _(ft) Elisa analysis: Interferon β (IFNB, IFNβ) Elisa wasperformed using either the IFNβ human ELISA Kit from Invitrogen or theHuman IFNβ ELISA Kit from PBL Interferon Source following themanufacturer's protocol.

Immunofluorescence Microscopy: Cells were cultured and treated in theirappropriate media on Lab-Tek II chamber slides. Cell were fixed with 4%paraformaldehyde for 15 minutes in at 37° C. and permeabilized with0.05% Triton X-100 for 5 minutes at room temperature. Immunostaining wasperformed with rabbit-anti-STING polyclonal followed by fluorescenceconjugated secondary antibodies (FITC-goat-anti-rabbit) (Invitrogen).Images were taken with Leica SP5 confocal microscope at the Image CoreFacility, University of Miami.

Northern blot analysis: Northern blot was performed with 51.ig of polyARNA using NorthernMax®-Gly Kit (Ambion). Briefly, RNA was resolved in 1%Glyoxal gels, transferred to the BrightStar®-Plus Nylon

Discussion

Demonstrated here is a protective role for STING in the prevention ofCAC induced by AOM/DSS carcinogenic treatment. Data indicates that thisevent may occur in large part through STINGS ability to control theproduction of IL-22BP. Following tissue damage, for example by DSS,IL-22 is induced and manifests protective, wound healing effects,including the promotion of tissue regeneration. However, if leftuncontrolled, IL-22 can also endorse tumor development. Thus, IL-22 istightly regulated by secreted IL-22BP, which is expressed by CD1 1c⁺dendritic cells. The importance of IL-22BP in controlling IL-22 has beenemphasized through observing that IL-22BP-deficient mice are alsosusceptible to AOM/DSS induced CAC, similar to STING-deficient mice.Nevertheless, IL-22 may have dual functions since mice lacking IL-22have also been reported to exhibit enhanced inflammatory responses whentreated repeatedly with DSS, plausibly because complete loss of IL-22may cause a delay in intestinal repair which in turn may actuallyaggravate inflammatory processes. The production of IL-22 BP can besuppressed by IL-18, which is known to be induced early afterDSS-induced intestinal damage. Accordingly, IL-18-deficient mice arealso susceptible to colon cancer, presumably through chronic suppressionof IL-22 activity, by unregulated IL-22BP, which may mimic the situationobserved with IL-22-deficient mice. Nevertheless, the control of IL-22BPremains to be fully clarified since down regulation of IL-22BP has alsobeen reported to occur in the absence of IL-18. In addition, it is knownthat loss of the TLR and IL-1R/IL-18R adaptor MyD88 also renders micesensitive to CAC, in part due to loss of IL-18R signaling. Finally,susceptibility to AOM/DDS-induced CAC has been shown to be enhanced inmice lacking Caspase-1, the adaptor PYCARD (Apoptosis-associatedspeck-like protein containing a CARD; ASC) or nucleotide-binding domain,leucine rich repeat and pyrin domain containing proteins 3 and 6(NLRP3/6), presumably since Pro-IL-18 produced by epithelial cells ordendritic cells requires cleavage prior to secretion into an activeform.

Data here indicates that IL-18 is inducible by dsDNA, or CDN's, or byAOM/DMH in a STING-dependent manner. Similar to the situation withIL-22, it is proposed that intestinal damage triggers STING activity (asa consequence of DNA damage or even from microbial ligands such as CDN'sor DNA). This results in the up-regulation of IL-18 and downregulationof IL-22BP, which would enable IL-22 to promote tissue repair. However,similar to the situation with IL-22, long term loss of STING may delaywound repair, facilitate microbial invasion trigger inflammation whichwould actually aggravate tumorigenesis. It was noted that IL-18expression was not totally ablated in tumors from SKO mice, presumablysince the expression of this cytokine could be induced by otherpathways. Despite this, IL-22BP levels remained low in SKO micedemonstrating the importance of STING in IL-22BP regulation.Collectively, the data indicates that STING plays a key role incontrolling intestinal tissue damage and CAC through regulatingIL-22BP's suppression of IL-22.

Given that loss of STING invokes a pro-tumorigenic state, at least inpart through an inability to transiently promote tissue repair or tosignal DNA damaging events to the immune system via secretion ofcytokines, the expression and function of STING in normal andcancer-related colon cells was explored. The study indicated that STINGwas expressed in the majority of colon cancer cells analyzed. However,it was observed that STING function was almost completely defective ingreater than 80% of the examined cells. Defects in STING signaling werealso observed in a wide variety of other tumor cells studied (FIG. 14).STING may associate with nucleic acids while CDN's are potentstimulators of STING activity. Cytoplasmic DNA can bind to the synthasecGAS and generate CDN's which then bind to and activate STING. Thisevent invokes STING trafficking with TBK1 via non-canonical autophagyprocesses, to endosomal regions harboring the transcription factors IRF3and NF-κB, resulting in cytokine activation. The data indicates thatSTING did not respond to transfected DNA and in many instances failed totranslocate. In these situations a lack of IRF3 activity andtranslocation was observed. Interestingly, loss of STING traffickingcoincided with a loss of cGAS expression (in greater than 30% of cases),presumably since CDN's were unavailable to facilitate STING function. Inother situations, defects in NF-κB activity were observed. Since bothNF-κB and IRF3 activity are required for the optimal production of typeI IFN and other cytokines, loss of either or both of these pathwayswould have detrimental effects on STING's ability to stimulate thetranscription of host defense genes, such as IL-18 or type I IFN,required for efficient anti-tumor T cells responses. It is proposed thatloss of STING signaling may enable DNA damaged cells to escape immunesurveillance and even promote inflammatory events due to an inability torepair damaged intestinal walls which may be vulnerable to invadingmicrobes.

Finally, it has been previously shown that STING plays a key role inprotecting against DNA virus infection. Since it was observed that STINGfunction was ablated in nearly all tumor cell-lines examined thus far,these cells' susceptibility to HSV1 and vaccinia virus (VV) infectionwas examined. The study indicated that colon cancer cells harboringdefects in STING function were highly sensitive to HSV1 and vacciniavirus infection. A number of oncolytic viruses, including HSV1, arebeing considered in the clinic as anti-tumor therapeutics, althoughunderstanding the mechanisms of action remain to be fully determined.The data here provides information on the causes of intestinaltumorigenesis and may provide prognostic information to dictate thesuccess of oncolytic viral therapy, and even disease outcome includingresponse to chemotherapeutic treatments.

Example 2

STING Function in Colorectal Adenocarcinoma

Defective STING signaling in colorectal adenocarcinoma cells:STING-deficient mice have been reported to be prone toAOM/DSS-associated CAC. However, whether STING function is deregulatedto any extent in human colorectal adenocarcinoma (CA) is unknown. Tostart to evaluate this, STING expression was examined by immunoblot in avariety of CA cells, generated from cancers diagnosed at various stagesas described by Duke's system. Results indicated that STING wasexpressed in 10 out of 11 cell lines examined, albeit at varying levels(FIG. 4A). To correlate expression levels with STING function, cellswere transfected with dsDNA to activate STING signaling, or with dsRNA(polyL⋅C) to activate the RIG-I like pathway. Type I IFN expression wasmeasured by ELISA, which is known to be STING-inducible. It was notedthat all 11 CA cells responded poorly to dsDNA-triggered type I IFNproduction (FIG. 4B). It was confirmed that all cells were transfectedadequately using FITC-labeled dsDNA activator and immunofluorescenceanalysis (FIG. 18). This was in contrast to control hTERT cells ornormal colon epithelial cells (FHC), which when transfected with dsDNAdid express IFNβ. In contrast, 8 of the 11 CA cells were able to producetype I IFN, in various amounts, in response to dsRNA, indicating thatthe RIG-I-Like pathway retained function in the majority of casesexamined (FIG. 4B). A similar finding was noted upon examination ofCXCL10 mRNA production by RT-PCR, although some CXCL10 was detected,albeit in low levels, in LoVo and HT29 in response to STING-dependentdsDNA transfection (FIG. 4D). To extend these findings further, IL-1 (3production was measured in the CA cells since it was previously notedthat carcinogen triggered DNA damage can induce IL-1 (3 throughSTING-signaling. Loss of IL-1 (3 has been shown to render micesusceptible to CAC due to wound healing responses being impaired. Thisstudy indicated that IL-10 was produced in the normal hTERT and FHCcells by dsDNA, indicating the importance of STING-activity in thisprocess. However, only 3 out of the 11 CA cells appeared able to produceIL-1 (3 in response to dsDNA treatment, again suggesting that STINGfunction is defective in the majority of CA cells examined (FIG. 4G).SW48, which lacked STING expression, did not appear responsive to dsDNAtransfection in any capacity. RNAi treatment confirmed that theupregulation of these cytokines was STING-dependent (FIGS. 19A-C). Giventhis data, a more detailed analysis of dsDNA-dependent STING signalingin CA cells was performed, by microarray analysis. CA cells wereselected based on their ability to exhibit some STING function or not.For example, data from FIG. 4D, indicated that HT29 and LoVo cells werepartially able to produce CXCL10 in response to dsDNA. In contrast,SW480 and HT116 were noted to be unable to produce CXCL10 to anysignificant level. Microarray analysis revealed that all the CA cellsexamined did not respond to dsDNA signaling as efficiently as controlFHC cells, and confirmed the RT-PCR analysis (FIGS. 4E, 4F). Forinstance, the level of CXCL10 was significantly higher in the controlFHC cells compared to the CA cells analyzed. However, HT29 cells didappear able to retain some response to cytosolic dsDNA, more than any ofthe other CA cells examined, especially when compared to SW480 or HT116(FIGS. 4E, F). While HT29 was able to produce IFNβ moderately asdetermined by microarray analysis, IFNβ protein production was notreadily evident by ELISA, perhaps due to low level expression, which wassimilarly observed even in the FHC controls (FIG. 4B). Nevertheless,taken together, the data indicates that a majority of CA cells exhibitdefective STING-dependent signaling with only SW1116, LS123, LoVo andHT29 exhibiting some low level activity.

Loss of IRF3 function in CA cells: To examine the extent of defectiveSTING signaling in CA cells, immunofluorescence and immunoblot analysiswas performed to evaluate NF-κB and IRF3 function. In the presence ofdsDNA, STING rapidly undergoes trafficking from the ER, along with TBK1,to perinuclear-associated endosomal regions, containing NF-κB and IRF3,in a process resembling autophagy (Ichikawa and Barber, 2008; Konno etal., 2013). This event accompanies STING phosphorylation anddegradation, likely to avoid sustained STING-activated cytokineproduction which can manifest inflammation. This approach confirmed thatSTING could traffic and undergo phosphorylation and degradation in thecontrol hTERT and FHC cells, following treatment with dsDNA (FIGS. 5Aand 5D, left panel). In these cells, TBK1 became phosphorylated as wellas its cognate target IRF3 and the p65 subunit of NF-κB (FIG. 5D, leftpanel). IRF3 and p65 were also noted to translocate into the nucleus, asexpected (FIGS. 5B, 5C). A comparable effect was observed using SW1116and LS123 CA cells which exhibited modest dsDNA-dependent IL-1 (3induction, confirming that the STING pathway retained some function inthese two cells (FIGS. 5A-D and FIGS. 4C, D). Similarly, HT29 and LoVodisplayed similar IRF3 translocation, but lacked p65 translocation. Thislikely explained that the defect in dsDNA-mediated innate immune geneinduction rested in the inability of STING to trigger p65 translocation(FIGS. 5A-D and FIGS. 4E,4F). However, it was noted that the other CAcells, such as SW480, SW1417, SW48 and HT116, exhibited very littleSTING activity or trafficking. Similarly, little evidence of TBK1 orIRF3 phosphorylation/translocation was noted. Some indication of p65phosphorylation was revealed, for example in SW480, but translocation ofthis transcription factor was not evident in any of these cells. STINGexpression was not observed in SW48 cells as previously described (FIGS.4A, 5A, 5D). This data indicates that dsDNA-signaling is affected atvarious points of the STING pathway. For example, STING retains someactivity and ability to traffic and escort TBK1 to IRF3, as in HT29 orLoVo cells, but NF-κB signaling is affected. In contrast, STING does notappear to undergo any phosphorylation or trafficking in SW480, SW1417,SW48 or HT116 cells, indicating that STING function is impeded upstreamof IRF3/NF-κB interaction.

CA cells exhibit defective cGAS expression: Loss of STING trafficking inSW480, SW1417, SW48 or HT116 cells could indicate a problem with STINGfunction in the ER, perhaps involving a mutation that would render STINGunable to interact with CDNs. Conversely, the breakdown inSTING-signaling could occur upstream and involve the synthase cGAS,which generates CDNs following association with dsDNA, to augment STINGfunction. To evaluate this, the entire STING genome within all 11 CAcells was sequenced (introns and exons comprise approximately 7.2 kb onchromosome 5q31.2). Sequence analysis indicated that 2 of the 11 CAcells (LoVo and SW480) exhibited a previously reported HAQ STING variant(Jin et al., 2011; Yi et al., 2013), which occurs in approximately 20%of the population, and which has been reported to be partially defectivewhen overexpressed in 293T cells, yet is able to function normally inthe presence of CDNs (FIG. 23). The remainder of the STING genesanalyzed represented the R272 encoded product, which has not beenreported to exert any defects in function and which representapproximately 85% of the population. Collectively, these findings do notsuggest the existence of a major mutation in the STING gene containedwithin the CA cells and suggest that a defect upstream of STING, forexample at the level of cGAS could plausibly be prevalent. We thusstarted to examine the expression and activity of cGAS in CA cells. AnRT-PCR assay was developed and principally measured cGAS mRNA levels.The results indicated that, of the 11 CA cells examined, cGAS expressionwas absent in 5 (55%) of them (LS174T, SW480, SW1417, SW48 and HT116)(FIG. 13A). This data was confirmed via immunoblot andimmunohistochemistry analysis using an antibody to cGAS (FIG. 13A, FIG.13C). A qPCR examination of 48 human colon adenocarcinoma samplessimilarly indicated low to undetectable level of cGAS in 15 of 48samples (31%) (FIG. 13B). Our findings could be explained through lossof the cGAS gene. However, sequencing analysis similarly indicated thatno major mutations or deletions existed within the genome encoding thecGAS gene (FIG. 24). In view of this, it was examined whether cGASexpression was suppressed by epigenetic phenomena, such as byhypermethylation of the cGAS promoter region (Lao and Grady, 2011;Mitchell et al., 2014). Indeed, databank analysis indicated the presenceof considerable CpG islands within the cGAS promoter region (FIG. 20A).Control hTERT, or cGAS-defective LS174T, SW480, SW1417, SW48 or HT116cells were thus treated with the de-methylating agent5-Aza-2′-deoxycytidine (SAZADC) for 5 days, and cGAS mRNA levels againevaluated. The study indicated that cGAS expression was rescued in 2 ofthe 5 cells examined (SW480 and HT116) (FIG. 13B). The sequencing ofbisulfite converted genomic DNA retrieved from normal and CA cellsconfirmed significant hypermethylation within the cGAS promoter regionof CA cells where cGAS expression is suppressed (FIG. 20B). It is notyet clear why expression levels of cGAS are muted in the remainder ofthe CA cells (LS174T, SW1417, SW48) but suppression could speculativelyinvolve other epigenetic modifications such as histone modifications(Jin and Robertson, 2013). Accordingly, treatment of these cells withhistone deacetylase or histone-lysine methyltransferases inhibitorspartially rescued cGAS mRNA expression in CA cells examined (FIG. 20C).It may also be apparent that alternate mechanisms of cGAS suppressionexist, such as those involving miRNAs. To determine if reconstitution ofcGAS expression rescued STING-dependent dsDNA signaling, control hTERTor SW480, HT116 (cGAS rescued by SAZADC) or LS174T (cGAS not rescued bySAZADC) CA cells were examined. It was observed that the SAZADC-treatedcGAS-rescued SW480, HT116 CA cells, but not LS174T cells regainedphosphorylation of TBK1 and IRF3, with concomitant phospho-IRF3translocation (FIG. 15C, D). These effects were reflected in modestexpression of type I IFN and IL-1β in the SAZADC treated SW480 and HT116CA cells (FIGS. 15E, F). Thus, demethylating agents may be able topartially rescue STING-dependent innate immune gene induction in selectCAs.

The question arises as to why the STING-signaling pathway may beinhibited in colon adenocarcinoma. Recently, it was shown thatSTING-deficient cells and mice are sensitive to AOM-induced DNA damage.In this situation, the STING pathway may play a role in the DNA-damageresponse pathway, to induce the production of cytokines which facilitatetissue repair or damaged cell removal (Chatzinikolaou et al., 2014;Kidane et al., 2014; Lord and Ashworth, 2012). As such, innate immuneinduction of CA cells in response to DNA damaging agents was examined.As shown in FIG. 21, the carcinogens AOM and DMH were able to induce theproduction of type I IFN in normal colon epithelial (CCD841) and inLS123 (which exhibited partial STING activity; FIGS. 4C and 4D).However, CA cells which exhibited defective STING activated IRF3 orNF-κB activity were unable to generate type I IFN in response to AOM orDMH. Thus, the inhibition of the STING pathway may enable DNA damagedcells, harboring mutations, to escape part of the DNA damage responseand the immune surveillance machinery to progress into a tumorigenicstate.

Tumors with defective STING-Signaling are sensitive to viral Oncolysis:The inventors have previously shown that loss of STING signaling invitro or in vivo renders cells or mice, respectively, extremelysensitive to Herpes simplex virus (HSV) infection. HSV, containing adsDNA genome of 375 kb is presently being evaluated in clinical trialsas a therapeutic agent for the treatment of cancer (Kolodkin-Gal et al.,2009). However, the mechanisms of oncolysis remain to be fullydetermined and there is no evaluation, presently, for determining theefficacy of HSV antitumor treatment. Given that it has been previouslyshown that STING signaling plays a critical role in host defenseresponses to HSV infection, and that STING activity is defective innumerous CA cells, it was postulated that the ability of STING to signalmay affect outcome to HSV therapy. To start to address this CA cells orcontrol hTERT and FHC were infected with HSV1 lacking the k34.5 encodingproduct that is presently being evaluated as an oncolytic agent,including against colon cancer as well as melanoma. The k34.5 viralprotein has been proposed to suppress host defense responses, althoughthe mechanisms remain to be fully clarified. Thus, HSV1k34.5 does notrobustly repress innate immune signaling events and potently triggersSTING-dependent innate immune gene induction, including type I IFNs.This analysis indicated that similar to our dsDNA transfection results,HSV1k34.5 induced the production of IFNβ mRNA significantly in controlhTERT and FHC cells, as well as SW1116 and LS123 CA cells (FIG. 16A).However, little type I IFN was induced in the remainder of the CA cells,including SW480 and HT116, deficient in cGAS expression. The ability toinduce type I IFN inversely correlated with HSV1k34.5 replication, dueto the induced anti-viral effects (FIG. 16B). Furthermore, cells such asSW480 and HT116 underwent rapid cell death, likely due to robust viralreplication, while control cells and cells with partial STING function(SW1116 and HT29) were significantly more refractory (FIG. 15C). Theexperiments were followed up by infecting CA cells with HSV expressingthe luciferase gene that contained k34.5 (HSV-Luc). This data confirmedthat CA cells exhibiting defective STING-signaling such as SW480 andHT116 enabled more viral-induced luciferase expression (FIG. 16D). siRNAtreatment further confirmed that the IFNβ responses induced by HSV1k34.5in normal and STING functional CA cells are STING dependent (FIG. 19D).Of note is that HSV1 is not the only DNA virus to be considered as anoncolytic therapeutic agent to treat cancer. Other candidate virusesunder consideration, including as a therapeutic against colon cancer,comprise Vaccinia Virus (VV), a dsDNA virus with 190 kb genome thatreplicates in the cytoplasm of infected cells. To evaluate whether VVcan trigger host innate immune response in the absence of functionalSTING signaling, we infected CA cells with partial STING signalingcapacity (SW116 and HT29) or completely defective STING signaling(SW480, HT116) with VV. Similar to the situation using HSV1k34.5, VVtriggered type I IFN and CXCL10 production only in the control cells orCA cells with partial STING signaling ability and not in cells with lossof STING function (SW480 and HT116) (FIGS. 16E, F). The data hereinindicates that CA cells with defective STING-signaling are highlysusceptible to HSV1 and VV oncolytic activity. Thus, it is plausiblethat being able to measure the presence or absence of STING/cGASexpression may help predict the response of patients with certaincancers to viral oncolytic therapy.

Predicting outcome to viral oncolytic therapy. The present dataindicates that the outcome of oncolytic virotherapy involving DNA-basedviruses such as HSV1 may be predicted by the presence or absence ofSTING/cGAS expression. Since the STING pathway naturally requires thepresence of STING and cGAS to function, and since it has been observedthat STING and/or cGAS may be absent in 30-55% of colon cancer, beingable to measure the presence of these two gene products may thereforeindicate the effectiveness of DNA-viral oncotherapy. This could beachieved using RT-PCR methodology but biopsied tissue may containinfiltrating hematopoietic cells that contain normal STING/cGASexpression. Thus, analysis of STING and/or cGAS protein or RNAexpression within the cancer cell itself would provide more accurateinformation into the status of STING function. Since an effectiveantibody to detect cGAS protein was not identified, a RNA in situhybridization assay was designed using RNAscope technology that candetect the single levels copies of an mRNA within individual cells. Bylabeling the STING probe green (FITC), and the cGAS probe red (Cy5),both probes were detectable in the same assay and the mRNA levels ofSTING and cGAS within the identical cell could be effectivelyquantitated. To test the assay, control cells or cGAS positive (SW1116or HT29) or negative (SW480 and HT116) CA cells were incubated with RNAprobes recognizing cGAS (red) or STING (green) mRNA. This studyindicated that STING and cGAS expression could be detected andquantitated in the control (hTERT and FHC) and STING/cGAS positive(SW1116 or HT29) CA cells using the RNAscope (FIGS. 17A, C). However,only STING was observed in the cGAS negative (SW480 and HT116) CA cells(FIGS. 17A, C). STING was not detectable in SW48 cells, as expected,using this assay (FIGS. 5A and 17A, C). This data also correlated withour previous expression analysis of cGAS in these cells by RT-PCR (FIG.13A). Moreover, cGAS expression was observed by RNAscope in those CAcells where cGAS mRNA production was rescued following treatment with5AZADC (SW480 and HT116); FIGS. 17B, D). Thus, fluorescence in situhybridization analysis may be able to predict the outcome to oncolyticviral therapy depending on the presence or absence of cGAS or STING.

To further follow up on this assay, normal hTERT, or cGAS positive(SW1116 or HT29) or negative (SW480 and HT116) CA cells were paraffinembedded, as well as SW48 which had both cGAS and STING expressionmissing. This situation may mimic situations where biopsied and paraffinembedded patient derived material required analysis. The experiment wasagain readily able to detect using the RNA probes both STING and cGASexpression in control, SW1116 and HT29 cells, as before, and only STINGin the cGAS negative SW480 and HT116 CA cells (FIGS. 17E, F). NeithercGAS nor STING was observed in the double negative SW48 line (FIGS. 4Aand 17E, F). This assay was further tested on 12 normal and 80 CAsamples in paraffin embedded tissue microarray (TMA) and it was observedthat STING was lost in 14% of CA samples and cGAS 15% of CA samples.Both STING and cGAS were lost in 9% of CA samples (FIGS. 17G, H).However, it was noted that STING expression and/or function was absentin a variety of other tumors, indicating that suppression of thispathway may be widespread in human cancer (FIG. 23). Thus, RNAscopeanalysis of STING/cGAS duel expression from paraffin embedded tissue mayhelp predict the outcome of select viral oncolytic therapy in vivo, asdetermined next.

In vivo analysis of CA cells with defective STING signaling toHSV11.34.5 therapy. To correlate the in vitro oncolytic effect of HSV1k34.5 in vivo, nude mice were subcutaneously inoculated with CA cellsharboring partial (SW1116 or HT29) or defective (SW480 and HT116) STINGsignaling. HSV1k34.5 was then administered intratumorally (FIG. 18A). Itwas observed that tumors exhibiting partial STING-signaling (SW1116 andHT29) were refractory to viral oncolytic treatment (FIGS. 18B, C).However, tumors derived from CA cells with defective STING-signalingwere noted to be extremely susceptible to virus treatment (FIGS. 18D,E). This data indicates that the activity of the STING pathway maypredict the outcome of HSV-related oncolytic therapy against colon aswell as other cancers.

Discussion

The STING controlled signaling pathway is essential for facilitatinginnate immune gene transcription in response to the recognition ofcytosolic DNA species. STING activity can be triggered by CDNs such ascyclic-di-AMP or cyclic-di-GMP produced from intracellular bacteria suchas Listeria monocytogenes or by cyclic-di-GMP-AMP (cGAMP) manufacturedby the synthase cGAS following association with cytosolic dsDNA species(Sun et al., 2013; Woodward et al., 2010). Such DNA can represent thegenome of DNA pathogens, such as HSV-1 or bacteria such as Mycobacteriumtuberculosis, as well as self DNA leaked from the nucleus of DNA damagedcells. STING-deficient mice, while viable, are extremely sensitive tolethal infection by a variety of pathogens. However, chronic STINGactivity has been shown to cause a diversity of autoinflammatorydisease, through the overproduction of pro-inflammatory cytokines.Indeed, inappropriate overstimulation of STING has even been shown toaggravate inflammation driven skin cancer (Ahn et al., 2014). However,transient STING activity has been shown to be essential for mediatingthe generation of anti-tumor T-cell responses. Data suggests that STING,in professional antigen presenting cells (CD8+ dendritic cells) becomesextrinsically activated by the DNA of engulfed dying tumor cells whichresults in the triggering of cytokines such as type I IFN, whichfacilitates cross-presentation and CTL priming. Correspondingly, thetherapeutic administration of CDNs, intratumorally, has been shown torepress tumor growth, presumably by facilitating DC-dependent CTLproduction (Corrales et al., 2015; Woo et al., 2014). STING may alsoplay a role in influencing the antitumor effects of checkpointinhibitors such as PD1, although the mechanisms remain to be determined.

The inventors have also recently demonstrated that STING-deficient miceare susceptible to carcinogen-aggravated CAC (Ahn et al., 2015). In thissituation, evidence indicates that damaged DNA can trigger STINGintrinsic activity, perhaps by leaking out of the nucleus or throughother mechanisms that remain to be clarified. Presumably, this eventwould augment cytokine production that would attract the immune systemto the damaged cell(s). Eradication of such cells may ensue, as well asthe stimulation of cytokine and growth factor dependent tissue repair.Data suggests that STING can trigger the production of cytokines thatfacilitate wound repair in the gut, such as IL-1β. Such cytokines areprocessed by nucleotide-binding oligomerization-domain protein likereceptors (NLRB) such as NLRP3 and NLRP6, which interact withinflammasome-associated ASC and caspase-1 to process IL-1 (3 and IL-18.These pro-inflammatory cytokines are secreted and bind to receptorsmainly requiring MyD88 for signaling. IL-18 production can suppressIL-22BP, which is responsible for inhibiting the wound repair activityof IL-22. Loss of ASC, caspase-I, MyD88 or IL22BP can increasetumorigenesis in colitis-associated colon cancer models, similar to lossof STING (Elinav et al., 2011; Huber et al., 2012; Salcedo et al.,2010). STING may thus work in concert with inflammasome processing.

Thus, loss of STING suppresses tissue healing and damaged mucosal liningmay enable the infiltration and expansion of bacteria with enhancedgenotoxic ability which would aggravate STING-independent inflammatoryresponses. The generation of ROS by overactive, infiltrating immunecells may enhance DNA damaging processes and facilitate mutationalinactivation of TSGs or the mutational activation of growth stimulatoryproteins such as k-ras. Thus, intrinsic STING-signaling may play a keyrole in preventing the development of cancer through responding to DNAdamage and alerting the immune surveillance machinery. In addition,extrinsic STING activity in DCs is also required for the generation ofanti-tumor CTLs. This places STING in a pivotal role in the hostanti-cancer defense arsenal.

Given this, the expression and regulation of STING signaling in coloncancer was analyzed and found frequent suppression of STING activity.These events inhibited the production of DNA-damage dependent cytokineproduction, which may enable the damaged cell to escape the attention ofthe immune surveillance system. Such cells may evade eradication andfurther genetic mutation events may accrue to enhance the tumorigenicprocess. The inhibition of STING signaling was observed to mainlyinvolve the suppression of STING expression, or of the synthase cGAS.Significant mutation or deletion events involving the STING or cGASgenes were not observed, but rather observed frequent transcriptionalsuppression involving hypermethylation of the promoter regions.Cytosolic DNA signaling was partially rescued using demethylating agentswhich regained cGAS expression in some but not other CA cells. However,it remains unclear whether the rescue of STING signaling in cancer cellsmay afford better responses to anti-cancer agents. Further, that cGASand in some cases STING expression was not rescued by demethylatingagents may indicate other forms of epigenetic silencing that requiresadditional characterization. In other CA types, it was observed that theability of STING to activate the transcription factors NF-κB or IRF3 wasimpaired, by molecular mechanisms that also remain to be determined. Itis noteworthy that a number of other genes involved in DNA repair, suchas the mismatch repair proteins MHS2 and MLH1 are also reported to befrequently silenced in colon cancer (Chatzinikolaou et al., 2014; Lordand Ashworth, 2012). Thus, targeting the DNA repair machinery maybe acommon requirement in cancer development. Collectively, it was observedthat STING-dependent signaling was defective in approximately 80% ormore of colon related tumors examined. This may indicate thatsuppression of STING function is also a key obligation for thetumorigenic process.

Since loss of STING may be common in tumors and may even predict outcometo anti-cancer therapy, the inventors developed assays to evaluate theexpression levels of both STING and cGAS. Loss of either of these twoproteins appears to repress cytosolic DNA mediated innate immunesignaling. The present ability to measure STING and cGAS mRNA expressionin situ, and STING protein expression using antibody enabled us todevelop a screen that indicated loss of one or other of these proteinsin over 40% of CAC. Such assays may be useful in predicting theeffective response rates of cancers to select therapeutic interventions.Further, recapitulating STING signaling in tumors, via novel antitumorgene therapy approaches, might enable such cells to reactivate hostantitumor immunity.

Accordingly, it was noticed that loss of STING signaling in CA cellsenabled the robust replication of DNA-based viruses such as HSV1.Viruses, such as HSV1 and vaccinia virus, are presently being used asoncolytic agents for the treatment of cancer. Such viruses may directlydestroy the tumor cell by lysis as well as create a tumor antigen sourcefor engulfment by APCs for the generation of CTLs. Data indicates thatSTING plays a key role in both these processes. However, the efficacy ofsuccessful oncoviral therapy remains low, for reasons that remainunclear. Mainly, assays based on molecular insight, that may helppredict treatment outcome have not been developed. This is because themolecular mechanisms that explain oncolysis in cancer cells rather thannormal cells remains to be fully appraised. Evidence suggests thatinnate immune signaling pathways that exert anti-viral activity may bedefective in cancer cells. Our data presented here is amongst the firstclear indication that loss of an innate signaling pathway can predictthe outcome to oncoviral therapy. Thus, utilization of molecularbiomarker assays similar to the ones portrayed here may enable a morepredictive response to the use of microbes for the treatment of cancer.Such assays may also shed insight into whether other STING-dependentanti-tumor therapies based on CDNs, or even DNA-adduct basedchemotherapeutic regimes, may work or not (Mansour, 2014; Rowe and Cen,2014). In this light, we have recently described that the immunologicalbenefits of using chemotherapeutic agents such as cisplatin andetoposide significantly involved the STING-signaling pathway. Thus,further studies on the regulation and function of STING in cancer mayprovide acumen into the molecular mechanisms of tumorigenesis as well asprovide a therapeutic target that may help in the treatment of cancer.

Experimental Procedures

Materials. All reagents were from Invitrogen, ThermoScientific or Sigmaunless specified.

Cell culture. Normal human cell and human cancer cell lines werepurchased from Lozna and ATCC respectively and cultured in theirappropriate growth media according to the instructions. Media andsupplements are from Invitrogen. hTERT-BJ1 Telomerase Fibroblasts(hTERT) were originally purchased from Clontech and were cultured in 4:1ratio of DMEM:Medium 199 supplement with 10% FBS, 4 mM L-Glutamine and 1mM sodium pyruvate at 37° C. in a 5% CO2-humidified atmosphere.

Immunoblot analysis. Equal amounts of proteins were resolved on sodiumdodecyl sulfate (SDS)-Polyacrylamide gels and then transferred topolyvinylidene fluoride (PVDF) membranes (Millipore). After blockingwith 5% Blocking Reagent, membranes were incubated with various primaryantibodies (and appropriate secondary antibodies). The image wasresolved using an enhanced chemiluminescence system ECL (ThermoScientific) and detected by autoradiography (Kodak). Antibodies: rabbitpoyclonal antibody against STING was developed in our laboratory asdescribed previously in Ishikawa et al, 2008; other antibodies wereobtained from following sources: β-actin (Sigma Aldrich), p-IRF3 (CellSignaling), IRF3 (Santa Cruz Biotechnology), p-p65 (Cell Signaling), p65(Cell Signaling), p-TBK1 (Cell Signaling), TB K1 (Abcam), cGAS (CellSignaling)

Interferon β Elisa analysis. Interferon β Elisa was performed usingeither the IFNβ human ELISA Kit from Invitrogen or the Human IFNβ ELISAKit from PBL InterferonSource following the manufacturer's protocol.

Immunofluorescence Microscopy. Cells were cultured and treated in theirappropriate media on Lab-Tek II chamber slides. Cell were fixed with 4%paraformaldehyde for 15 minutes in at 37° C. and permeabilized with0.05% Triton X-100 for 5 minutes at room temperature. Immunostaining wasperformed with rabbit-anti-STING polyclonal, rabbit-anti-IRF3 (SantaCruz Biotechnology) or rabbit-anti-p65 (Cell Signaling) followed byfluorescence conjugated secondary antibodies (FITC-goat-anti-rabbit)(Invitrogen). Images were taken with Leika LSM confocal microscope atthe Image Core Facility, University of Miami.

Microarray Analysis. Total RNA was isolated from cells or tissues withRNeasy Mini kit (Qiagen). RNA quality was analyzed by Bionalyzer RNA6000 Nano (Agilent Technologies). Gene array analysis was examined byIllumina Sentrix BeadChip Array (Human HT-12 V4 Bead Chip) at theOncogenomics Core Facility, University of Miami. Gene expressionprofiles were processed and statistical analysis was performed at theBioinformatics Core Facility, University of Miami. Briefly, rawintensity values from 11lumina array are uploaded on GeneSpring™software from Agilent. Values are Quantile normalized and log 2transformed to the median of all samples. Significantly differentialexpressed genes from a two-class comparison are computed using theStudent's t-test and selected using threshold of P-value <0.05.Hierarchical Clustering and visualization of selected differentiallyexpressed genes is performed on GeneSpring using Pearson Correlationdistance method and linkage was computed using the Ward method. FoldChange analysis was performed between two groups and differentiallyexpressed genes were selected based on threshold of Fold Changes.

Quantitative Real-Time PCR (qPCR). Total RNA was reverse transcribedusing QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR wasperformed with the TaqMan gene Expression Assay (Applied Biosystems).

Immunohistochemistry and Histological Analysis. Tissue Microarray waspurchased from Origene. Immunohistochemistry staining was performed withrabbit-anti-cGAS antibody following standard protocol.

HSV1γ34.5 Amplification, Purification, Titration and Infection.HSV1γ34.5 was amplified in Vero cells and purified by sucrose gradientultracentrifugation following standard protocol. Plague assay usingserial diluted virus was performed in Vero cells following standardprotocol. Cells were infected with HSV1γ34.5 at specific M.O.I. for 1hour, washed and then incubated for designated period for specific assayexamination.

RNA in situ Hybridization. STING and cGAS RNA probed was custom designedby ACD and RNA in situ Hybridization was performed using RNAscope®Multiplex Fluorescent Reagent Kit for cultured cells and 2-plexRNAscope® Reagent Kit for FFPE cells and tissue following themanufacturer's instruction.

Mouse Treatment. Balb/C nu/nu mice were purchased from Charles River.Tumor cells were introduced in the flanks of Balb/c nude mice bysubcutaneous injection of 2E106 of the appropriate tumor cells andtumors allowed to develop to an average diameter of approximately 0.5cm. HSV1γ34.5 was then be injected into the tumors every other day for atotal of three times at appropriate dosage (i.e. 50 μl at 1E7). PBS wasused as vehicle control. Effects on tumor growth was monitored. Micewere euthanized when tumor diameter exceeds 10 mm.

Bisulfite Sequencing Analysis. Bisulfite conversion of genomic DNA wasperformed using EZDNA Methylation Kit from Zymo Research followed by PCRamplification. PCR products were then gel purified and sequenced.

Statistical Analysis. All statistical analysis was performed byStudent's t test unless specified. The data were considered to besignificantly different when P<0.05.

Example 3 STING Function in Melanoma

Given the findings above, the studies were extended into evaluatingSTING function in melanoma, in part because such cancers appear to besusceptible to viral oncolytic treatment, which suggests defects ininnate immune pathways. Here it is reported that STING mediated innateimmune signaling is largely impaired both in human melanoma derivedcells and in primary patient melanoma-derived tissues. Loss of STINGand/or cGAS expression in melanoma was recurrently found, predominantlythrough epigenetic hypermethylation silencing. These findings suggestthat suppression of STING signaling may be an important part of tumordevelopment. Moreover, loss of STING function rendered melanoma cellsmore susceptible to HSV1 and vaccinia virus-mediated oncolysis.Therefore, the development of a prognostic assay that enables themeasurement of STING or cGAS expression may lead to a better indicationof the efficacy of viral oncolytic treatment.

Recurrent loss of STING signaling in human melanoma derived cell lines.The STING-controlled innate immune pathway has been reported to belargely impaired in human colon cancers, an event which may facilitatetumorigenesis (Xia T, Konno H, Ahn J, Barber G N. Deregulation of STINGSignaling in Colorectal Carcinoma Constrains DNA Damage Responses andCorrelates With Tumorigenesis. Cell reports. 2016; 14:282-97). Toevaluate whether this key pathway is similarly defective in other typesof cancer STING expression was further examined by immunoblot in a panelof human malignant melanomas. These results showed that STING expressionwas not detectable in 3 out of 11 cell lines examined (G361, MeWo andSK-MEL-5) and STING expression level was dramatically suppressed in afurther 3 cell lines (SK-MEL-2, SK-MEL-28 and WM115) (FIG. 26A). Thesynthase cGAS resides upstream of STING and generates CDN's capable oftriggering STING function. Next, the expression of cGAS by immunoblotwas examined and it was found that this synthase was absent in 4 out of11 cell lines examined (A375, G361, SK-MEL-5 and SK-MEL-24) (FIG. 26A).Real-time PCR analysis using cGAS probe confirmed that cGAS was notdetectable in A375 and SK-MEL-5, but low level of cGAS was detected inG361 and SK-MEL-24 (FIG. 26A). To correlate STING/cGAS expressionanalysis with functional STING signaling, cells were transfected withdsDNA to activate STING-dependent cytokine production, or with dsRNA(polyL⋅C) to activate the STING-independent RIG-I like signaling pathwayand measured type I IFN expression by ELISA (Ichikawa et al., 2008).This study indicated that all 11 melanoma cells responded poorly toSTING-dependent, dsDNA-triggered type I IFN production. Usingfluorescence microscopy analysis, it was confirmed that all cells wereindeed transfected with FITC-labeled dsDNA activator. However, controlhTERT cells and normal human melanocytes (HEMa) were able to expresshigh levels of IFNβ when transfected with dsDNA, suggesting the STINGmediated type I interferon responses were suppressed specifically in themelanoma cells (FIG. 26B). This finding was further supported byreal-time PCR analysis, in which dsDNA stimulated IFNβ and CXCL10induction was suppressed in majority of the melanoma cells examined,although weak activity were detected in SK-MEL-24 and SK-MEL-31 cells(FIG. 26C-D). In contrast, 6 of the 11 Melanoma cells were able toproduce type I IFN and CXCL10, albeit at various levels, in response todsRNA, indicating that the RIG-I-Like RNA signal pathway were mostlyintact in majority of melanoma cells examined (FIG. 26C-D). Using siRNAtreatment to knock down STING expression in normal cells and 2 melanomascell-lines (SK-MEL-24, SK-MEL31) that appeared to retain partial STINGactivity, it was confirmed that the upregulation of these dsDNA-inducedcytokines was STING-dependent. Taken together, our data indicates thatSTING-dependent signaling is largely impaired in a majority of melanomacells with only SK-MEL-24 and SK-MEL-31 exhibiting weak STING activity.

Loss of STING dependent TBK1-IRF3 activation in Melanoma cells. Toexamine the extent of STING signaling defect in melanoma cells, IRF3 andNF-κB activation were evaluated by immunofluorescence microscopy andimmunoblot analysis. When stimulated with dsDNA, STING rapidly undergoestranslocation from the ER, along with TBK1, to perinuclear-associatedendosomal regions, containing NF-κB and IRF3, in a procedure similar toautophagy (Ichikawa et al., 2008, Konno et al., 2013). This incidentaccompanies STING phosphorylation and degradation, almost certainly toavoid prolonged STING-induced cytokine production which is now known toprovoke chronic inflammation (Ahn et al., 2014). Our results confirmedthat, following dsDNA treatment in normal hTERT cells, STINGtranslocated to perinuclear region and underwent phosphorylation anddegradation events, (FIGS. 27A and 27D, left panel). During thisprocess, TBK1 was phosphorylated in hTERT cells as well as its cognatetarget IRF3 and the p65 subunit of NF-κB (FIG. 27D). IRF3 and p65translocation into the nucleus was also observed, indicating normalactivation (FIGS. 27B, C and D). A similar effect was observed inSK-MEL-24 and SK-MEL-31 cells which exhibited partial dsDNA-dependentcytokine production, confirming that these two cell lines retained someSTING function (FIG. 27A-D and FIG. 26B-D). However, while RPMI7951 andSK-MEL-3 retained STING/cGAS expression and displayed similar IRF3activation upon dsDNA treatment, these cells lacked p65 translocation.This observation would explain why dsDNA failed to trigger type I IFNproduction, which requires both IRF3 and NF-κB for its transcriptionalactivation (FIG. 27A-D and FIG. 26B-D). In addition, in cells whereSTING and/or cGAS expression were not detected (such as A375, G361, MeWoand SK-MEL-5), no evidence of TBK1 or IRF3 phosphorylation/translocationwas detected in these cells following dsDNA treatment (highlighted byboxes) (FIG. 27B, D). Although phosphorylated p65 was observed, notranslocation of this transcription factor into the nucleus was evidentin any of the RPMI7951, SK-MEL-3, A375, G361, MeWo or SK-MEL-5 cells(FIG. 27C-D). These results indicate that dsDNA induced STING signalingis deregulated at various points along the pathway in many of themelanoma cell lines examined. For example, while STING retained somesignaling activity and ability to induce the translocation of IRF3, asin RPMI7951 and SK-MEL-3 cells, NF-κB signaling was observed to beaffected. In contrast, STING did not appear to undergo anyphosphorylation or translocation in A375, G361, MeWo or SK-MEL-5 cells,suggesting that STING function is affected upstream of IRF3/NF-κBactivation, likely due to loss of STING and/or cGAS expression.

RNAscope and IHC analysis of STING/cGAS expression. Since the STINGpathway requires the presence of STING and cGAS, and since STING and/orcGAS expression was observed to be absent in—40% melanoma cellsexamined, being able to measure the presence of STING and cGAS could beuseful in predicting functional STING signaling in melanoma. Althoughimmunoblot and RT-PCR methodology is effective in examining STING/cGASexpression in cultured cell lines, biopsied tissue often contains notonly tumor cells but also other cell types including infiltrating immunecells that could retain normal STING/cGAS expression (Ichikawa et al.,2009). Thus, analysis of STING and/or cGAS protein or RNA expressionwithin the cancer cell itself is necessary for accurate evaluation intothe presence of these products. As described above, an RNA in situhybridization assay using RNAscope technology that can efficientlydetect STING/cGAS mRNA copies within individual cells. By usingFITC-labelled STING probe (green), and Cy5-labelled cGAS probe (red),melanoma cells were examined using RNA fluorescent in situ hybridization(RNA FISH). Results showed that both probes combined within the sameassay effectively detected STING and cGAS mRNA in control HEMa cells.STING mRNA was also detected in A375, SK-MEL-24 and SK-MEL-31 cells butnot in G361, MeWo or SK-MEL-5 cells whereas cGAS mRNA was not detectedin A375 or SK-MEL-5 cells (FIG. 28A). mRNA copy numbers were quantitatedwith results being consistent with our previous results obtained usingour expression analysis (FIG. 26A, 28A). Thus, RNA fluorescence in situhybridization analysis can effectively quantitate STING/cGAS expressionsimultaneously in single cells.

mRNA expression by chromogenic in situ hybridization (RNA CISH) ofparaffin embedded melanoma cells was also evaluated. This situation maymimic situations where biopsied and paraffin embedded patient derivedmaterial are generally used for biomarker analysis. This study was ableto detect and quantitate both STING and cGAS mRNA expression inSK-MEL-24 and SK-MEL-31 cells as before. In A375 cells, only STING wasdetected whereas cGAS was absent. STING was not detected in G361 or MeWocells. Both STING and cGAS were absent in SK-MEL-5 cells (FIG. 28B).Overall RNA CISH analysis generated similar results to RNA FISHevaluation.

Using antibody to cGAS and STING, immunohistochemistry (IHC) analysis onparaffin embedded cells was also performed, which confirmed cGAS andSTING protein expression status in accord with our immunoblot andRNAscope studies (FIG. 28C).

IHC analysis of STING/cGAS expression in Melanoma TMA. To evaluateSTING/cGAS expression in patient-derived melanoma samples, wesubsequently examined by IHC analysis a paraffin embedded melanomatissue microarray (TMA, MEL961, Pantomics) that contains 8 normal skintissues, 8 benign nevus tissues, 56 malignant melanoma tissues and 24metastatic melanoma tissues. It was observed that all normal tissuesexpressed both STING and cGAS. cGAS was not detected in 2 benign nevustissues, while STING was noted to be present in all 8 nevi. In malignantmelanoma tissues, 23.2% of melanoma samples lost STING expression, while16.1% of melanoma samples did not express cGAS, and both STING and cGASwere absent in 14.3% of melanoma tissues. In more advanced metastaticmelanoma tissue, loss of both STING and cGAS was more profound (41.7%)(FIG. 29). Given this data, suppression of STING or cGAS expression maycommonly occur in human melanoma and plausibly other human cancers (Xiaet al, 2016). In summary, our IHC procedures may be useful for theanalysis of cGAS and STING expression in FFPE preserved clinical tumorsamples.

STING/cGAS expression may be suppressed through DNA hypermethylation inmelanoma cells. Loss of STING/cGAS expression could occur through eithergenetic alteration or mutation. To evaluate the gene status of STING andcGAS in melanoma cells, we sequenced the STING and cGAS gene within all11 melanoma cells. Sequence analysis of the entire STING gene (intronsand exons comprise approximately 7.2 kb on chromosome 5q31.2) indicatedthat 7 of the 11 melanoma cells exhibited HAQ STING variant (Jin et al.,2011, Yi et al., PloS One, 2013), which was previously reported to occurin approximately 20% of the population. STING gene in all melanoma cellsas well as normal HEMa cell contains the R272 polymorphism, which wasreported to represent approximately 85% of the population but does notexert any defects in STING function. Collectively, sequence analysis didnot reveal any major genetic defect in the STING gene within themelanoma cells. Similar sequence analysis was also carried out on cGASexons. However, no major mutations or deletions were noted. Takentogether, genetic mutations or deletions do not seem to be involved inSTING/cGAS defective expression in melanoma cells.

In view of this, it was examined whether STING or cGAS expression wassuppressed by epigenetic processes, such as by hypermethylation of thepromoter regions (20, 21). Indeed, databank analysis indicated thepresence of considerable CpG islands within the STING and cGAS promoterregion. As such, melanoma cells lacking either STING or cGAS expressionwere treated with the de-methylating agent 5-Aza-2′-deoxycytidine(SAZADC) for 5 days and evaluated recapitulation of STING or cGASexpression. Real-time PCR analysis showed that cGAS expression wasrecovered in A375 cells as well as SK-MEL-5 cells although at lowerextent. Although SK-MEL-24 exhibited low cGAS expression by RT-PCR,SAZADC treatment did not seem to affect cGAS expression level ofSK-MEL-24 cells significantly (FIG. 30A). This result was againconfirmed by both immunoblot and RNA FISH analysis, in which cGASexpression was apparently recapitulated in A375 and SK-MEL-5 cellsfollowing SAZADC demethylation (FIG. 30B-C). In MeWo cells, STINGexpression was restored by 5AZADC treatment as shown by both immunoblotand RNA FISH analysis. However, STING remained absent in similarlytreated G361 cells as well as in SK-MEL-5 cells, although cGASexpression was partially restored in the same treated SK-MEL-5 cells(FIG. 30A-C). Therefore DNA hypermethylation is involved in silencingSTING or cGAS expression in some melanoma cells (A375 and MeWo). Howeverit is not yet clear why expression levels of STING are muted in theremainder melanoma cells (G361, SK-MEL-5). Other epigeneticmodifications such as histone modifications or other transcriptionregulator factors such as miRNA could be involved in suppressing STINGand/or cGAS expression (Jin et al., 2013, Yarbrough et al., 2014). Todetermine if reconstitution of STING/cGAS expression rescuedSTING-dependent dsDNA signaling, IFNβ and CXCL10 induction was examinedin 5AZADC treated melanoma cells following dsDNA stimulation. Inductionof both IFNβ and CXCL10 production was observed in cGAS rescued A375cells, as well as modest expression of IFNβ in STING rescued MeWo cells,concomitant with IRF3 and STING translocation (FIG. 30D-G). Whereas noSTING function was observed in G361 or SK-MEL-5 cells following 5AZADCtreatment, confirmed that both STING and cGAS are necessary for dsDNAstimulated cytokine production (FIG. 30D-E). Thus, demethylating agentsmay be able to partially rescue STING-dependent innate immune geneinduction in select melanoma cells.

Defect in STING signal renders melanoma cells susceptible to DNA virusinfection. STING innate immune signaling plays a critical role in hostdefense responses to DNA viruses. For example, mice lacking STING areextremely sensitive to Herpes simplex virus (HSV) infection (Ishikawa etal., 2008, Ishikawa et al., 2009). A strain of HSV1 lacking the γ34.5gene, referred to as talimogene laherparepvec (OncoVex, T-VEC) ispresently being evaluated in clinical trials as a therapeutic agent forthe treatment of cancer including melanoma (Andtbacka et al., 2015;Lawler et al., 2015; Kolodkin-Gal et al., 2009). However, the mechanismsof oncolysis remain to be fully determined and there is no evaluation,presently, for determining the likely efficacy of HSV-based antitumortreatment. Is was previously shown that STING activity is defective innumerous colon cancer cells which renders cells sensitive to DNA virusinfection including HSV1. We postulated that lack of STING function inmelanomas cells may correlate with an increased susceptibility to DNAvirus infection and replication. Plausibly, the ability of STING toeffectively signal may affect outcome to HSV-based oncoviral therapy. Tostart addressing this we infected the melanoma cells or control hTERTand HEMa with HSV1 lacking the γ34.5 gene similar to the strainpresently being investigated as an oncolytic agent against humanmelanoma. The γ34.5 viral protein has been proposed to suppress hostdefense responses, although the mechanisms need to be fully clarified.Thus, without the robust repression of the host innate immune signaling,HSV1γ34.5 is able to potently trigger STING-dependent innate immuneactivation, including type I IFN production (Ichikawa et al., 2009).Similar to dsDNA treatment, HSV1γ34.5 induced robust production of IFNβand CXC110 mRNAs in control hTERT and HEMa cells, as well as inSK-MEL-24 and SK-MEL-31 cells that retained partial STING signaling(FIG. 31A-B). However, little type I IFN production was observed in theremainder of the melanoma cells. Loss of the ability to induce type IIFN correlated with increased HSV1γ34.5 replication, likely due to theimpaired anti-viral effects, especially in melanoma cells lackingSTING/cGAS expression (A375, G361, MeWo and SK-MEL-5) (FIG. 31C).Furthermore, cells with defective STING signal underwent rapid celldeath, likely due to robust viral replication whereas control cells andcells with partial STING function (SK-MEL-24 and SK-MEL-31) weresignificantly more resistant (FIG. 31D). This data confirmed thatmelanoma cells exhibiting defective STING-signaling enabled more HSV1replication and lysis.

The ability of Vaccinia Virus (VV) to activate host innate immunesignaling in the absence of STING function in melanoma cells was alsoexamined. VV, a dsDNA virus with 190 kb genome that replicates in thecytoplasm of infected cells, is another candidate DNA virus that iscurrently under evaluation as an oncolytic therapeutic agent to treatcancer (Rowe et al., 2014). Similar to our observations using HSV1γ34.5,VV triggered type I IFN and CXCL10 production only in the control cellsand melanoma cells with partial STING function but not in cells withloss of STING/cGAS expression (A375, G361, MeWo and SK-MEL-5). Ourresults indicate that melanoma cells with defective STING-signaling arehighly susceptible to HSV1 and VV infection. Thus, it is plausible thatmelanoma lacking STING/cGAS expression are more sensitive to DNA virusoncolytic activity and being able to measure STING/cGAS expression inmelanoma tissue may help predict the response of patients to selectedviral oncolytic therapy.

In vivo analysis of melanoma cells to HSV1γ34.5 therapy. Our in vitroanalysis indicated that loss of STING signaling may affect the outcomeof select oncoviral therapy (FIG. 31A-D). To further evaluate thispossibility, in vivo, melanoma xenografts were generated bysubcutaneously inoculating nude mice with melanoma cells harboringpartial (RPMI7951 and SK-MEL-3) or defective (A375, MeWo and SK-MEL-5)STING signaling. HSV1γ34.5 was then administered intratumorally andtumor growth monitored (FIG. 32). Results showed that tumors derivedfrom melanoma cells with defective STING-signaling were extremelysusceptible to HSV1γ34.5 treatment (FIG. 32A-B). Tumor size decreasedrapidly after HSV1γ34.5 treatment. 4 out of 6 A375 tumors and 3 out of 5SK-MEL-5 tumors diminished 2-3 weeks after treatment (FIG. 32A-B). Incontrast tumors derived from melanoma cells exhibiting partial STINGsignaling (RPMI7951 and SK-MEL-3) were refractory to viral oncolytictreatment (FIG. 32C-D). While these tumors are slow growing in vivo,majority of mice did not respond to HSV1γ34.5 therapy at all and theanimals were sacked after the tumor burden became significant.Therefore, our findings complement our previous studies and indicatethat the ability of measure STING function in melanoma may predict theoutcome of DNA virus-related oncolytic therapy against human melanomaand perhaps other type of cancers.

Discussion

As reported above, STING signaling is frequently suppressed in humancolon cancer. As mentioned, loss of intrinsic STING signal may play akey role in preventing cancer development through inability to respondto DNA damage and alert the immune surveillance machinery(Chatzinikolaou et al., 2014, Kondo et al., 2013). To extend thesestudies, the expression and regulation of STING signaling in melanomawas analyzed and it was similarly found that STING-dependent cytokineproduction was frequently suppressed in human melanoma. Although nosignificant mutation or deletion events involving the STING or cGASgenes was observed, the inhibition of STING signaling was found tomainly occur through epigenetic suppression of STING and or cGASexpression. Cytosolic DNA mediated STING signaling was partially rescuedby demethylating agent (5AZADC) treatment in some STING-defectivemelanoma cells, suggesting DNA hypermethylation is one of the mechanismsfor STING/cGAS suppression. However, in other STING-defective melanomacells, demethylation was not effective in being able to restore STINGexpression. STING and/or cGAS may selectively become targets forsuppression at various stages of cancer development, the suppression ofeither being sufficient to impede STING function. It was also noticed insome melanoma cells, that although both STING/cGAS were expressed, theability of STING to effectively activate the transcription factors NF-κBor IRF3 was impaired by molecular mechanisms that remain to bedetermined. Thus, STING function can be impaired at different stepsalong the signaling pathway, although epigenetic suppression of eitherSTING/cGAS expression seems to be common. Collectively, it was observedthat STING-dependent signaling was defective in numerous melanomas whichindicated that inhibiting STING function maybe a key obligation for thedevelopment of melanoma, plausibly enabling such cells to evade theimmune system.

Loss of STING may be common in tumors and may even predict outcomes toanti-cancer therapy. Accordingly, assays were developed herein toevaluate the expression levels of both STING and cGAS, loss of either ofwhich will affect STING function. These assays were validated inmelanoma and showed that both RNAish based and IHC based assays wereable to measure STING and cGAS mRNA or protein expression in melanomacells accurately and sensitively. Using IHC, a melanoma TMA was screenedwhich showed loss of either STING or cGAS in over 50% malignant and over60% metastatic melanomas. Loss of STING function may not be a key tumoronset factor. However, STING does appear to be important in thegeneration of cytokines in response to DNA damage (Ahn et al., 2015, Xiaet al., 2016, Ahn et al., 2014). Loss of STING function is almostcertainly important in later stages of cancer development to escapeimmunosurveillance and host anti-tumor immunity, especially beneficialin tumor metastasis. The assays described may be useful in predictingthe effective response rates of cancers to select therapeuticinterventions. Furthermore, recapitulating STING signal in tumors, vianovel antitumor gene therapy approaches, may reactivate host antitumorimmunity against escaped tumor cells.

Accordingly, it was noticed that loss of STING function in melanomacells rendered cells highly sensitive to DNA-virus mediated oncolyticeffect (such as HSV1). Oncolytic HSV1 is one viral therapeutic agent inclinical application. For example, talimogene laherparepvec (T-VEC)(Amgen) is a herpes simplex virus type 1 (HSV-1) based OV that has beenengineered to express granulocyte-macrophage colony-stimulating factor(GM-CSF) to increase immune recognition. Although T-VEC has shownimproved effect over traditional immune therapies for advanced melanoma,the overall response rate is still limited. This phenomena could bepotentially due to diverse STING/cGAS expression status among melanomacases. Oncolytic viruses may directly destroy the tumor cell by lysis aswell as create a tumor antigen source for activation of anti-tumorimmune response (Woo et al., 2015). STING may play key roles in both ofthese processes. Therefore, utilization of STING/cGAS as molecularbiomarker may enable a more predictive response to the use of microbesfor the treatment of cancer. Such assays may also shed insight into theefficacy of other STING-dependent anti-tumor therapies based on CDNs, oreven DNA-adduct based chemotherapeutic regimes (Zitvogel et al., 2013).Further, gene therapies involving modification of the STING/cGAS statusmay provide advantages of utilizing host innate and adaptive defensemechanism to facilitate antitumor effects in combination withtraditional anti-tumor therapies. Thus, further studies on STING signalin cancer development may provide insight into the molecular mechanismsof human carcinogenesis as well as provide novel anti-tumor therapeuticapproaches.

Experimental Procedure

Materials. All reagents were from ThermoFisher Scientific or Sigmaunless specified.

Cell culture. Normal human melanocytes (HEMa) and human melanoma celllines were purchased from ThermoFisher Scientific and ATCC respectivelyand cultured in their appropriate growth media according to theinstructions. hTERT-BJ1 Telomerase Fibroblasts (hTERT) were originallypurchased from Clontech and were cultured in 4:1 ratio of DMEM:Medium199 supplement with 10% FBS, 4 mM L-Glutamine and 1 mM sodium pyruvateat 37° C. in a 5% CO2-humidified atmosphere.

Immunoblot analysis. Equal amounts of proteins were resolved on sodiumdodecyl sulfate (SDS)-Polyacrylamide gels and then transferred topolyvinylidene fluoride (PVDF) membranes (Millipore). After blockingwith 5% Blocking Reagent, membranes were incubated with various primaryantibodies (and appropriate secondary antibodies). The image wasresolved using an enhanced chemiluminescence system ECL (ThermoScientific) and detected by autoradiography (Kodak). Antibodies: rabbitpoyclonal antibody against STING was developed in our laboratory asdescribed previously in Ishikawa et al, 2008; other antibodies wereobtained from following sources: β-actin (Sigma Aldrich), p-IRF3 (CellSignaling), IRF3 (Santa Cruz Biotechnology), p-p65 (Cell Signaling), p65(Cell Signaling), p-TBK1 (Cell Signaling), TB K1 (Abcam), cGAS (CellSignaling).

Interferon β Elisa analysis. Interferon β Elisa was performed as above.

Immunofluorescence Microscopy. Cells were cultured and treated in theirappropriate media on Lab-Tek II chamber slides. Cell were fixed with 4%paraformaldehyde for 15 minutes in at 37° C. and permeabilized with0.05% Triton X-100 for 5 minutes at room temperature. Immunostaining wasperformed with rabbit-anti-STING polyclonal, rabbit-anti-IRF3 (SantaCruz Biotechnology) or rabbit-anti-p65 (Cell Signaling) followed byfluorescence conjugated secondary antibodies (FITC-goat-anti-rabbit).Images were taken with Leika LSM confocal microscope at the Image CoreFacility, University of Miami.

Quantitative Real-Time PCR (qPCR). Total RNA was reverse-transcribedusing QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR wasperformed with the TaqMan gene Expression Assay (Applied Biosystems).

Immunohistochemistry and Histological Analysis. Tissue Microarray waspurchased from Pantomics. Immunohistochemistry staining was performedwith rabbit-anti-cGAS antibody or rabbit-anti-STING antibody followingstandard protocol.

Virus Amplification, Purification, Titration and Infection. HSV-1 γ34.5was kindly provided by Bernard Roizman. Vaccinia virus (vTF7-3) waskindly provided by John Rose. Virus was amplified in Vero cells andpurified by sucrose gradient ultracentrifugation following standardprotocol. Plague assay using serial diluted virus was performed in Verocells following standard protocol. Cells were infected with virus atspecific M.O.I. for 1 hour, washed and then incubated for designatedperiod for specific assay examination.

RNA in situ Hybridization. STING and cGAS RNA probed was custom designedby ACD and RNA in situ Hybridization was performed using RNAscope®Multiplex Fluorescent Reagent Kit for cultured cells and 2-plexRNAscope® Reagent Kit for FFPE cells and tissue following themanufacturer's instruction.

Mouse Treatment. Balb/C nu/nu mice were purchased from Charles River andmaintained in the institutional Division of Veterinary Resources (DVR).All experiments were performed with institutional animal care and usecommittee (IACUC) approval and in compliance with IACUC guidelines.Tumor cells were introduced in the flanks of Balb/c nude mice bysubcutaneous injection of 2E106 of the appropriate tumor cells andtumors allowed to develop to an average diameter of approximately 0.5cm. HSV1γ34.5 was then be injected into the tumors every other day for atotal of three times at 1E7PFU. PBS was used as vehicle control. Effectson tumor growth were monitored. Mice were euthanized when tumor diameterexceeds 10 mm.

Genomic DNA Sequencing. Genomic DNA was extracted from melanoma cells aswell as normal cells using Qiagen DNeasy Kit and specific locus wassequenced by Polymorphic DNA Technologies.

Statistical Analysis. All statistical analysis was performed byStudent's t test unless specified. The data were considered to besignificantly different when P<0.05.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed:
 1. A method for treating a cancer with an oncolyticvirus: a) performing a PCR assay on a sample from a mammal to determineif the mammal has a defective functional activity of Stimulator ofInterferon Genes (STING) in a cell population in the sample; b) if themammal has a defective functional activity of STING, then identifying aselected therapy, where the selected therapy is administering one ormore oncolytic viruses having a double stranded (ds) deoxynucleic acid(DNA) (dsDNA) genome selected from the group consisting of herpessimplex virus, Varicella Zoster virus, and vaccinia virus; and c)internally treating the mammal with the selected therapy.
 2. The methodof claim 1, where the functional activity of STING is determined to bedefective based at least in part on the amount of cyclic GuanosineMonophosphate (cGMP)—Adenosine Monophosphate (AMP) Synthase (cGAS) inthe cell population.
 3. The method of claim 2, where the functionalactivity of STING is determined to be defective when an immune responsein the mammal is enhanced by administration of the one or more oncolyticviruses are selected from the group consisting of herpes simplex virus-1γ₁34.5 deletion and vaccinia virus vTF7-3.
 4. The method of claim 3,where the immune response includes modulation of T cell activity,modulation of dendritic cell activity, and modulation of immunecytokines.
 5. The method of claim 2, further comprising where thefunctional activity of STING is determined to be defective based atleast in part on determining a cellular level of one or more mRNA'sselected from the group consisting of Interleukin-1 (IL-1), IL-3, IL-18and IL-22 in the cell population.
 6. The method of claim 5, where thecancer is a colorectal adenocarcinoma and the selected therapy comprisesadministering to the subject the oncolytic virus HSV1k34.5, where theoncolytic virus induces the production of interferons in the mammal. 7.The method of claim 1, further comprising where the functional activityof STING is determined to be defective based at least in part ondetermining the cellular level of interleukin-22 binding proteins in thecell population.
 8. The method of claim 7, further comprising where thefunctional activity of STING is determined to be defective based atleast in part on determining the cellular level of Interleukin-1 (IL-1),IL-18 and IL-22 in the cell population.
 9. The method of claim 1, wherethe selected therapy is administration of dsDNA in combination withinterferon-beta.
 10. The method of claim 1, further comprising if thesample does not have the defective functional activity of STING,administering a cancer treatment to the subject that does not cause aDNA mutation.
 11. The method of claim 1, where the cancer iscolitis-associated cancer.
 12. The method of claim 1, where an immuneresponse in the cancer that has the defective functional activity ofSTING is enhanced by administration of the one or more oncolytic virusesselected from the group consisting of herpes simplex virus-1 γ₁34.5deletion and vaccinia virus vTF7-3.
 13. The method of claim 1, where thetherapy results in increased tumor cell death and/or retarded tumorgrowth.
 14. The method of claim 1, where the sample is selected from thegroup consisting of a body fluid, a cell sample, a tissue sample, abiopsy sample, a tissue print, a skin sample, and a hair sample.
 15. Themethod of claim 14, where the body fluid is selected from the groupconsisting of blood, urine, plasma, saliva, or cerebrospinal fluid. 16.The method of claim 1, where the sample is a soluble fraction of a cellpreparation, where the cells are grown in cell culture in vitro and themedia from the cell culture is tested for functional activity of STING.17. A method for treating a cancer comprising: a) determining whether amammal having one or more of colorectal cancer, colon cancer andmelanoma cancer has a defective functional activity of cyclic GuanosineMonophosphate (cGMP)—Adenosine Monophosphate (AMP) Synthase (cGAS) byperforming a PCR assay on a sample of the mammal to determine a cellularlevels of one or more mRNA's selected from the group consisting ofInterleukin-1 (IL-1), IL-3, IL-18 and IL-22 to determine if a cellpopulation has a defective functional activity of cGAS; b) if the mammalhas the defective functional activity of cGAS, then selecting a therapyfor the cancer, where the therapy involves administering one or moreoncolytic viruses having a double stranded (ds) deoxynucleic acid (DNA)(dsDNA) genome selected from the group consisting of herpes simplexvirus and vaccinia virus, and c) administering the selected therapy tothe mammal.
 18. The method of claim 17, where the functional activity ofcGAS is defective if an immune response in the mammal is enhanced byadministration of the one or more oncolytic viruses are selected fromthe group consisting of herpes simplex virus-1 γ₁34.5 deletion andvaccinia virus vTF7-3.
 19. A method for treating a cancer comprising: a)determining whether a mammal having one or more of colorectal cancer,colon cancer and melanoma cancer that has failed at least onechemotherapy regimen has a defective functional activity of cyclicGuanosine Monophosphate (cGMP)—Adenosine Monophosphate (AMP) Synthase(cGAS) measured by performing a first PCR assay on a first sample of themammal to determine if one or more mRNA's selected from the groupconsisting of Interleukin-1 (IL-1), IL-3, IL-18 and IL-22 is lowered; b)if the mammal has the defective functional activity of cGAS, thenperforming a first oncolytic therapy of administering one or moreoncolytic viruses selected from the group consisting of herpes simplexvirus-1 γ₁34.5 deletion and vaccinia virus vTF7-3; c) performing asecond PCR assay on a second sample of the mammal to determine if one ormore mRNA's selected from the group consisting of IL-1, IL-3, IL-18 andIL-22 remains lowered to determine whether the mammal continues to havea defective functional activity of cGAS; and d) i) if the mammalcontinues to have the defective functional activity of cGAS, thenperforming a second oncolytic therapy comprising administering one ormore oncolytic viruses selected from the group consisting of herpessimplex virus, Varicella Zoster virus, and vaccinia virus; or ii) if themammal has normal cellular levels of IL-1, IL-3, IL-18 and IL-22 mRNA's,then performing a second oncolytic therapy comprising administering asecond agent that can increase cGAS levels in the subject.
 20. Themethod of claim 19, where in step d) i) the one or more oncolyticviruses are administered in conjunction with a third agent.